Design, synthesis, and biological evaluation of dinaciclib and CAN508 hybrids as CDK inhibitors.

The scaffolds of two known CDK inhibitors (CAN508 and dinaciclib) were the starting point for synthesizing two series of pyarazolo[1,5-a]pyrimidines to obtain potent inhibitors with proper selectivity. The study presented four promising compounds; 10d, 10e, 16a, and 16c based on cytotoxic studies. Compound 16a revealed superior activity in the preliminary anticancer screening with GI % = 79.02-99.13 against 15 cancer cell lines at 10 µM from NCI full panel 60 cancer cell lines and was then selected for further investigation. Furthermore, the four compounds revealed good safety profile toward the normal cell lines WI-38. These four compounds were subjected to CDK inhibitory activity against four different isoforms. All of them showed potent inhibition against CDK5/P25 and CDK9/CYCLINT. Compound 10d revealed the best activity against CDK5/P25 (IC50 = 0.063 µM) with proper selectivity index against CDK1 and CDK2. Compound 16c exhibited the highest inhibitory activity against CDK9/CYCLINT (IC50 = 0.074 µM) with good selectivity index against other isoforms. Finally, docking simulations were performed for compounds 10e and 16c accompanied by molecular dynamic simulations to understand their behavior in the active site of the two CDKs with respect to both CAN508 and dinaciclib.

In Vitro Evaluation of Oxidative Stress Induced by Oxime Reactivators of Acetylcholinesterase in HepG2 Cells.

Oxime reactivators of acetylcholinesterase (AChE) are used as causal antidotes for intended and unintended poisoning by organophosphate nerve agents and pesticides. Despite all efforts to develop new AChE reactivators, none of these drug candidates replaced conventional clinically used oximes. In addition to the therapeutic efficacy, determining the safety profile is crucial in preclinical drug evaluation. The exact mechanism of oxime toxicity and the structure-toxicity relationship are subjects of ongoing research, with oxidative stress proposed as a possible mechanism. In the present study, we investigated four promising bispyridinium oxime AChE reactivators, K048, K074, K075, and K203, and their ability to induce oxidative stress in vitro. Cultured human hepatoma cells were exposed to oximes at concentrations corresponding to their IC50 values determined by the MTT assay after 24 h. Their potency to generate reactive oxygen species, interfere with the thiol antioxidant system, and induce lipid peroxidation was evaluated at 1, 4, and 24 h of exposure. Reactivators without a double bond in the four-carbon linker, K048 and K074, showed a greater potential to induce oxidative stress compared with K075 and K203, which contain a double bond. Unlike oximes with a three-carbon-long linker, the number of aldoxime groups attached to the pyridinium moieties does not determine the oxidative stress induction for K048, K074, K075, and K203 oximes. In conclusion, our results emphasize that the structure of oximes plays a critical role in inducing oxidative stress, and this relationship does not correlate with their cytotoxicity expressed as the IC50 value. However, it is important to note that oxidative stress cannot be disregarded as a potential contributor to the side effects associated with oximes.

Discovery of (E)-2-(hydroxyimino)-N-(2 ((4methylpentyl)amino)ethyl)acetamide (KR-27425) as a non-pyridinium oxime reactivator of paraoxon-inhibited acetylcholinesterase.

This study aimed to explore non-pyridinium oxime acetylcholinesterase (AChE) reactivators that could hold the potential to overcome the limitations of the currently available compounds used in the clinic to treat the neurologic manifestations induced by intoxication with organophosphorus agents. Fifteen compounds with various non-pyridinium oxime moieties were evaluated for AChE activity at different concentrations, including aldoximes, ketoximes, and α-ketoaldoximes. The therapeutic potential of the oxime compounds was evaluated by assessing their ability to reactivate AChE inhibited by paraoxon. Among the tested compounds, α-Ketoaldoxime derivative 13 showed the highest reactivation (%) reaching 67 % and 60 % AChE reactivation when evaluated against OP-inhibited electric eel AChE at concentrations of 1,000 and 100 µM, respectively. Compound 13 showed a comparable reactivation ability of AChE (60 %) compared to that of pralidoxime (56 %) at concentrations of 100 µM. Molecular docking simulation of the most active compounds 12 and 13 was conducted to predict the binding mode of the reactivation of electric eel AChE. As a result, a non-pyridinium oxime moiety 13, is a potential reactivator of OP-inhibited AChE and is taken as a lead compound for the development of novel AChE reactivators with enhanced capacity to freely cross the blood-brain barrier.

Sustainable ionic liquids-based molecular platforms for designing acetylcholinesterase reactivators.

We report a green chemistry approach for preparation of oxime-functionalized ILs as AChE reactivators: amide/ester linked IL, l-alanine, and l-phenylalanine derived salts bearing pyridinium aldoxime moiety. The reactivation capacities of the novel oximes were evaluated towards AChE inhibited by typical toxic organophosphates, sarin (GB), VX, and paraoxon (PON). The studied compounds are mostly non-toxic up to the highest concentrations screened (2 mM) towards Gram-negative and Gram-positive bacteria cell lines and both filamentous fungi and yeasts in the in vitro screening experiments as well as towards the eukaryotic cell (CHO-K1 cell line). Introduction of the oxime moiety in initially biodegradable structure decreases its ability to biodegradation. The compound 3d was shown to reveal remarkable activity against the AChE inhibited by VX, exceeding conventional reactivators 2-PAM and obidoxime. The regularities on antidotal activity, cell viability, plasma stability, biodegradability as well as molecular docking study of the newly synthesized oximes will be used for further improvement of their structures.

Multi-Responsive Molecular Encapsulation and Release Based on Hydrogen-Bonded Azo-Macrocycle.

Research on stimuli-responsive host-guest systems is at the cutting edge of supramolecular chemistry, owing to their numerous potential applications such as catalysis, molecular machines, and drug delivery. Herein, we present a multi-responsive host-guest system comprising azo-macrocycle 1 and 4,4'-bipyridinium salt G1 for pH-, photo-, and cation- responsiveness. Previously, we reported a novel hydrogen-bonded azo-macrocycle 1. The size of this host can be controlled through light-induced E↔Z photo-isomerization of the constituent azo-benzenes. The host is found in this work to be capable of forming stable complexes with bipyridinium/pyridinium salts, and implementing guest capture and release with G1 under light in a controlled manner. The binding and release of the guest in the complexes can also be easily controlled reversibly by using acid and base. Moreover, the cation competition-induced dissociation of the complex 1a2⊃G1 is achieved. These findings are expected to be useful in regulating encapsulation for sophisticated supramolecular systems.

A Specific Interaction between Ionic Liquids' Cations and Reichardt's Dye.

Solvatochromic probes are often used to understand solvation environments at the molecular scale. In the case of ionic liquids constituted by an anion and a cation, which are designed and paired in order to obtain a low melting point and other desirable physicochemical properties, these two indivisible components can interact in a very different way with the probe. This is the case with one of the most common probes: Reichardt's Dye. In the cases where the positive charge of the cation is delocalized on an aromatic ring such as imidazolium, the antibonding orbitals of the positively charged aromatic system are very similar in nature and energy to the LUMO of Reichardt's Dye. This leads to an interesting, specific cation-probe interaction that can be used to elucidate the nature of the ionic liquids' cations. Parallel computational and experimental investigations have been conducted to elucidate the nature of this interaction with respect to the molecular structure of the cation.

Retinal Pigment Epithelium in Human Donor Eyes Contains Higher Levels of Bisretinoids Including A2E in Periphery than Macula.

Purpose: With age, human retinal pigment epithelium (RPE) accumulates bisretinoid fluorophores that may impact cellular function and contribute to age-related macular degeneration (AMD). Bisretinoids are comprised of a central pyridinium, dihydropyridinium, or cyclohexadiene ring. The pyridinium bisretinoid A2E has been extensively studied, and its quantity in the macula has been questioned. Age-changes and distributions of other bisretinoids are not well characterized. We measured levels of three bisretinoids and oxidized A2E in macula and periphery in human donor eyes of different ages. Methods: Eyes (N = 139 donors, 61 women and 78 men, aged 40-80 years) were dissected into 8 mm diameter macular and temporal periphery punches. Using liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS) and an authentic synthesized standard, we quantified A2E (ng). Using LC-ESI-MS and a 50-eye-extract of A2E, we semiquantified A2E and 3 other compounds (eye extract equivalent units [EEEUs): A2-glycerophosphoethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE), and monofuranA2E (MFA2E). Results: A2E quantities in ng and EEEUs were highly correlated (r = 0.97, P < 0.001). From 262 eyes, 5 to 9-fold higher levels were observed in the peripheral retina than in the macula for all assayed compounds. A2E, A2DHPE, and MFA2E increased with age, whereas A2GPE remained unaffected. No significant right-left or male-female differences were detected. Conclusions: Significantly higher levels were observed in the periphery than in the macula for all assayed compounds signifying biologic differences between these regions. Levels of oxidized A2E parallel native A2E and not the distribution of retinal illuminance. Data will assist with the interpretion of clinical trial outcomes of agents targeting bisretinoid-related pathways.

The Negative Solvatochromism of Reichardt's Dye B30 - A Complementary Study.

The UV/Vis spectra of a hypothetical negative solvatochromic dye in a solvent are theoretically calculated assuming the classical damped harmonic oscillator model and the Lorentz-Lorenz relation. For the simulations, the oscillator strength of the solvent was varied, while for the solute all oscillator parameters were kept constant. As a result, a simple change of the oscillator strength of the solute can explain the redshift and intensity increase of the UV/Vis band of the solute. Simulated results are compared with measured UV/Vis spectroscopic data of 2,6-diphenyl-4-(2,4,6-triphenylpyridinium-1-yl) phenolate B30 (Reichardt's dye) Significant correlations of the absorption energy (1/λmax ) with the molar absorption coefficient ϵ as function of solvent polarity are demonstrated for several derivatives of B30. The approach presented is only applicable to negative solvatochromism. Therefore, while the approach is vital to fully understand solvatochromism, it needs to be complemented by other approaches, e. g., to describe the changes of the chemical interactions based on the nature of the solvent, to explain all its various aspects.

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

Charged pyridinium oximes with thiocarboxamide moiety are equally or less effective reactivators of organophosphate-inhibited cholinesterases compared to analogous carboxamides.

The organophosphorus antidotes, so-called oximes, are able to restore the enzymatic function of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) via cleavage of organophosphate from the active site of the phosphylated enzyme. In this work, the charged pyridinium oximes containing thiocarboxamide moiety were designed, prepared and tested. Their stability and pKa properties were found to be analogous to parent carboxamides (K027, K048 and K203). The inhibitory ability of thiocarboxamides was found in low µM levels for AChE and high µM levels for BChE. Their reactivation properties were screened on human recombinant AChE and BChE inhibited by nerve agent surrogates and paraoxon. One thiocarboxamide was able to effectively restore function of NEMP- and NEDPA-AChE, whereas two thiocarboxamides were able to reactivate BChE inhibited by all tested organophosphates. These results were confirmed by reactivation kinetics, where thiocarboxamides were proved to be effective, but less potent reactivators if compared to carboxamides.

Cooperative Weak Dispersive Interactions Actuate Catalysis in a Shape-Selective Abiological Racemase.

A simple abiological host-guest system demonstrates racemase activity with catalytic rate enhancements of 104 without employing traditional functional groups. Cooperative weak interactions enhanced through shape-complementarity between the catalyst active site and the reaction transition state drive this activity, as proposed by Pauling for enzymes. In analogy to the Jencks' concept of catalytic antibodies, it is shown that a hapten resembling the planar transition state of the bowl inversion acts as a potent inhibitor of this catalytic process. In contrast, no substrate/product inhibition is detected, and a relatively weak binding of the substrate is observed (Ka ≈ 102 M-1 at 293 K). This simple box-and-bowl system demonstrates that shape selectivity arising from cooperative dispersive forces suffices for the emergence of a catalytic system with an enzyme-like thermodynamic profile.

A dye encapsulated zinc-based metal-organic framework as a dual-emission sensor for highly sensitive detection of antibiotics.

Self-assembly of two Zn-MOFs, [Zn2L(DMF)3]·H2O·5DMF (1) and [Zn2L(H2O)2]·4H2O·3DMF (2), was achieved with an amide-functionalized tetracarboxylate ligand under similar conditions. Incorporated amide groups make the tetratopic linkers exhibit different configurations, tetrahedron and square, and subsequently combine tetrahedral [Zn2(CO2)4] clusters or square paddle-well [Zn2(CO2)4] clusters to afford a lon net for 1 and a nbo net for 2. Remarkably, 2 demonstrated high porosity and amide group decorated cages, and thereby proved to be a good capturing agent for a fluorescent dye molecule (DMASM). Consequently, a dual-emitting DMASM@2 sensor was successfully fabricated based on effective energy transfer from the host framework to DMASM with the variable luminescent color being visible to the naked eye. DMASM@2 could be used for the detection of metronidazole (MDZ) and dimetridazole (DTZ) with high sensitivity and remarkable recyclability.

Anticancer activity of N-heteroaryl acetic acid salts against breast cancer; in silico and in vitro investigation.

BACKGROUND: The present research was performed to assess N-heteroaryl acetic acid salts' anticancer activity against the breast cancer cell in order to introduce new inhibitory agents for histone deacetylase. METHODS AND RESULTS: A molecular docking simulation was performed to design the rational novel compounds. Afterward, the best compounds were selected for synthesis. The cytotoxic effects and mechanism of action have been studied via (Methyl Thiazol-Tetrazolium) MTT assay. Flow cytometry and gene expression analyses were performed to introduce an effective acetic acid derivative as an anticancer agent. Molecular docking simulations demonstrated that all compounds have the best interaction with histone deacetylase. The fold changes of Bcl-2, Bak, Bim, Caspase-3, and Caspase-8 gene expressions were investigated and compared with reference gene using real-time PCR. The cytotoxic studies showed the best anticancer activity of 4-benzyl-1-(carboxymethyl) pyridinium bromide (compound 2) with a low IC50 value (32 µM, p < 0.05). Also, the best anti HDAC activity was obtained for compound 2 with IC50 value of 1.1 µM. Furthermore, this compound showed a high percentage of apoptosis among all tested compounds after 72 h incubation which was associated with the significant increase in mRNA level of Bim, Bax, Bak, Caspase-3, and Caspase-8 and the considerable decrease in Bcl2 gene expression. CONCLUSION: These results suggest that compound 2 with the benzyl ring could be an effective anticancer compound for further investigation in breast cancer treatment.

Visible-light-mediated catalyst-free synthesis of unnatural α-amino acids and peptide macrocycles.

The visible light induced, photocatalysts or photoabsorbing EDA complexes mediated cleavage of pyridinium C-N bond were reported in the past years. Here, we report an ionic compound promote homolytic cleavage of pyridinium C-N bond by exploiting the photonic energy from visible light. This finding is successfully applied in deaminative hydroalkylation of a series of alkenes including naturally occurring dehydroalanine, which provides an efficient way to prepare ß-alkyl substituted unnatural amino acids under mild and photocatalyst-free conditions. Importantly, by using this protocol, the deaminative cyclization of peptide backbone N-terminals is realized. Furthermore, the use of Et3N or PPh3 as reductants and H2O as hydrogen atom source is a practical advantage. We anticipate that our protocol will be useful in peptide synthesis and modern peptide drug discovery.

A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes.

Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and their unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. Here, we present a structurally precise chemoproteomics probe for the biologically active molecule 20(S)-hydroxycholesterol (20(S)-OHC) and provide a map of its proteome-wide targets in the membranes of living cells. Our target catalog consolidates diverse OHC ontologies and demonstrates that OHC-interacting proteins cluster with specific processes in immune response and cancer. Competition experiments reveal that 20(S)-OHC is a chemo-, regio- and stereoselective ligand for the protein transmembrane protein 97 (Tmem97/the σ2 receptor), enabling us to reconstruct the 20(S)-OHC-Tmem97 binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of metabolite activity and identify actionable targets for molecular therapy.

Interest of novel N-alkylpyridinium-indolizine hybrids in the field of Alzheimer's disease: Synthesis, characterization and evaluation of antioxidant activity, cholinesterase inhibition, and amyloid fibrillation interference.

A small library of molecules combining indolizine and N-alkyl pyridinium was synthesized and evaluated in a multi-target-directed-ligand strategy for Alzheimer's disease (AD) treatment. The new compounds were classified in three series depending on the number of methylene residues linking the two heterocycles (Ind-PyCx with x = 0, 2 or 3). The molecules were synthesized from the corresponding bis-pyridines by two-step formation of the indolizine core including mono-alkylation of pyridine and 1,3-dipolar cycloaddition with an alkylpropiolate. Their activities against AD's key-targets were evaluated in vitro: acetyl- and butyrylcholinesterase (AChE and BChE) inhibition, antioxidant properties and inhibition of amyloid fibril formation. None of the three series showed significant activities against all the targets. The Ind-PyC2 and Ind-PyC3 series are active on eeAChE and hAChE (µM IC50 values). Most of the positively charged molecules from these two series also appeared active against eqBChE, however they lost their activity on hBChE. Comparative molecular modeling of 13 and 15 docked in hAChE and hBChE highlighted the importance of the substituent (p-methoxybenzoyl or methyloxycarbonyl, respectively) located on the indolizine C-3 for the binding. The larger molecule 13 fits more tightly at the active site of the two enzymes than 15 that shows a larger degree of freedom. The Ind-PyC2 and Ind-PyC3 hybrids displayed some antioxidant activity when tested at 750 µg/mL (up to 95% inhibition of DPPH radical scavenging for 10). In both series, most hybrids were also able to interact with amyloid fibers, even if the inhibitory effect was observed at a high 100 µM concentration. The Ind-PyC0 molecules stand out completely due to their spectroscopic properties which prevent their evaluation by Ellman's and ThT assays. However, these molecules showed interesting features in the presence of preformed fibers. In particular, the strong increase in fluorescence of 3 in the presence of amyloid fibers is very promising for its use as a fibrillation fluorescent reporter dye.

Novel Dipyridinium Lipophile-Based Ionic Liquids Tethering Hydrazone Linkage: Design, Synthesis and Antitumorigenic Study.

Novel dicationic pyridinium ionic liquids tethering amphiphilic long alkyl side chains and fluorinated counter anions have been successfully synthesized by means of the quaternization of the dipyridinium hydrazone through its alkylation with different alkyl halides. The resulting halogenated di-ionic liquids underwent a metathesis reaction in order to incorporate some fluorinated counter anions in their structures. The structures of all the resulting di-ionic liquids were characterized by several spectroscopic experiments. The antitumorigenic activities of the investigated compounds were further studied against three different human lung cancer cell lines. Compared to the standard chemotherapeutic agent, cisplatin, the synthesized di-ionic liquids exerted equal, even more active, moderate, or weak anticancer activities against the various lung cancer cell lines under investigation. The observed anticancer activity appears to be enhanced by increasing the length of the aliphatic side chains. Moreover, dicationic pyridinium bearing a nine carbon chain as counter cation and hexafluoro phosphate and/or tetrafluoro bororate as counter anion were selected for further evaluation and demonstrated effective and significant antimetastatic effects and suppressed the colonization ability of the lung cancer cells, suggesting a therapeutic potential for the synthesized compounds in lung cancer treatment.

Pd(II) complexes with chelating N-(1-alkylpyridin-4(1H)-ylidene)amide (PYA) ligands: Synthesis, characterization and evaluation of anticancer activity.

The bidentate N-(1-Alkylpyridin-4(1H)-ylidene)amide (PYA) pro-ligands [H2LBn][Cl]2 (2), and [H2LMe][TfO]2 (3) were prepared by simple alkylation reactions of the known compound, N,N-di(pyridin-4-yl)oxalamide (H2L, 1). The Pd(II) complexes, [Pd(LBn)2][Cl]2 (4), [Pd(LMe)2][Cl][TfO] (5), Pd(LBn)Cl2 (6) and Pd(LMe)Cl2 (7) were synthesized through reactions between these pro-ligands and suitable Pd(II) substrates in the presence of base. The molecular structures of 3 and 6 were obtained by single crystal X-ray structure determinations. Studies of the experimental and computational DNA binding interactions of the compounds 1-7 revealed that overall 4 and 6 have the largest values for the binding parameters Kb and ΔGbo. The results showed a good correlation with the steric and electronic parameters obtained by quantitative structure activity relationship (QSAR) studies. In-vitro cytotoxicity studies against four different cell lines showed that the human breast cancer cell lines MCF-7, T47D and cervical cancer cell line HeLa had either higher or similar sensitivities towards 4, 6 and 2, respectively, compared to cisplatin. In general, the cytotoxicity of the compounds, represented by IC50 values, decreased in the order 4 > 6 > 2 > 5 > 3 > 1 > 7 in cancer cell lines. Apoptosis contributed significantly to the cytotoxic effects of these anticancer agents as evaluated by apoptosis studies.

Gated Dissipative Dynamic Artificial Photosynthetic Model Systems.

Gated dissipative artificial photosynthetic systems modeling dynamically modulated environmental effects on the photosynthetic apparatus are presented. Two photochemical systems composed of a supramolecular duplex scaffold, a photosensitizer-functionalized strand (photosensitizer is Zn(II)protoporphyrin IX, Zn(II)PPIX, or pyrene), an electron acceptor bipyridinium (V2+)-modified strand, and a nicking enzyme (Nt.BbvCI) act as functional assemblies driving transient photosynthetic-like processes. In the presence of a fuel strand, the transient electron transfer quenching of the photosensitizers, in each of the photochemical systems, is activated. In the presence of a sacrificial electron donor (mercaptoethanol) and continuous irradiation, the resulting electron transfer process in the Zn(II)PPIX/V2+ photochemical module leads to the transient accumulation and depletion of the bipyridinium radical-cation (V·+) product, and in the presence of ferredoxin-NADP+ reductase and NADP+, to the kinetically modulated photosynthesis of NADPH. By subjecting the mixture of two photochemical modules to one of two inhibitors, the gated transient photoinduced electron transfer in the two modules is demonstrated. Such gated dissipative process highlights its potential as an important pathway to protect artificial photosynthetic module against overdose of irradiance and to minimize photodamage.

Deaminative Reductive Methylation of Alkylpyridinium Salts.

Methyl groups can imbue valuable properties in organic molecules, often leading to enhanced bioactivity. To enable efficient installation of methyl groups on simple building blocks and in late-stage functionalization, a nickel-catalyzed reductive coupling of secondary Katritzky alkylpyridinium salts with methyl iodide was developed. When coupled with formation of the pyridinium salt from an alkyl amine, this method allows amino groups to be readily transformed to methyl groups with broad functional group and heterocycle tolerance.