Compounds 3a-c Research Articles

Pyrimethamine conjugated histone deacetylase inhibitors: Design, synthesis and evidence for triple negative breast cancer selective cytotoxicity

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor which has been recognized as a promising cancer therapeutic target. Small molecule pyrimethamine (PYM) is a known direct inhibitor of activated STAT3 and it is currently under clinical trial. Also, histone deacetylase (HDAC) inhibition has been shown to indirectly attenuate STAT3 signaling through inhibition of STAT3 activation. Herein we described the design and biological profiling of two classes of PYM-conjugated HDAC inhibitors (HDACi). We observed that the class I PYM-HDACi compounds 12a-c potently inhibited HDACs 1 and 6 in cell free assays while a lead class II PYM-HDACi compound 23 showed a strong HDAC 6 selective inhibition. In a cell-based assay, 12a-c are preferentially cytotoxic to MDA-MB-231, a TNBC cell line that is highly STAT3-dependent, while 23 showed no such selective toxicity. Subsequent target validation studies revealed that a representative class I PYM-HDACi compound 12c elicited a signature of HDAC and STAT3 pathway inhibition intracellularly. Collectively, these data suggest that PYM-HDACi compounds are promising leads to develop targeted therapy for TNBC.

Synthesis, Characteristic and Antimicrobial Activity of Some New Spiro[indol-thiazolidon-2,4-diones] and Bis(5-fluorospiro[indoline-3,2’-thiazolidine]-2,4’-dione) Probes

In a search for new antimicrobial agents, some new spiro[indol-thiazolidon- 2,4-diones] (6a-c) were synthesized by condensation of 5-substituted isatins 1 with sulfanilamide in MeOH, followed by aroylation with p-nitrobenzoyl chloride in DMF to get compounds 4a-c. Cycloaddition of 4a-c with thioglycolic acid in a dry non-polar solvent (dioxane) gave the targets 6a-c. Also, bis(5-fluorospiro[indoline-3,2’-thiazolidine]-2,4’-dione) (9) was synthesized by condensation of 5-fluoroindoline-2,3-dione with benzene-1,4-diamine (2:1 by mol) in MeOH, which followed by cycloaddition with thioglycolic acid in dioxane gave compound 8. Acylation of the later with 2,2,2-trifluoroacetic anhydride in THF has yielded the target 9. Structures of the products have been deduced from their elemental analysis and spectral data. The in vitro antimicrobial activity of the new systems 6a-c, and 9 was tested.

Synthesis, characterization, and biological evaluation of a set of new alkylthio substituted plastoquinones containing ester group

In the present investigation, fourteen new alkylthio substituted 5,6-dimethyl-1,4-benzoquinone derivatives were synthesized as potential antimicrobial and antifungal agents. First, 2,3-dichloro-5,6-dimethyl-1,4-benzoquinone was reacted with alkyl thiols containing ester group to yield its mono- and bis(thio) derivatives and then these compounds were investigated for their antibacterial and antifungal activities. In vitro studies were carried out using four Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 4352, Proteus mirabilis ATCC 14153), three Gram-positive bacteria (Enterococcus faecalis ATCC 29212, Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 29213), and two fungi (Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019) by using the microbroth dilutions technique. Most of the synthesized compounds (3a-c, 3f, 4b-c, and 4e) exhibited good antibacterial activities with MIC values between 9.76 and 78.12 μg/mL. Compound 4e was identified as a hit compound with antibacterial efficiency against P. aeruginosa and E. faecalis with MIC value of 9.76 and 19.53 μg/mL, respectively.

Benzoxazole/benzothiazole-derived VEGFR-2 inhibitors: Design, synthesis, molecular docking, and anticancer evaluations.

A novel series of benzoxazole/benzothiazole derivatives 4a-c-11a-e were designed, synthesized, and evaluated for anticancer activity against HepG2, HCT-116, and MCF-7 cells. HCT-116 was the most sensitive cell line to the influence of the new derivatives. In particular, compound 4c was found to be the most potent derivative against HepG2, HCT-116, and MCF-7 cells, with IC50 values = 9.45 ± 0.8, 5.76 ± 0.4, and 7.36 ± 0.5 µM, respectively. Compounds 4b, 9f, and 9c showed the highest anticancer activities against HepG2 cells with IC50 values of 9.97 ± 0.8, 9.99 ± 0.8, and 11.02 ± 1.0 µM, respectively, HCT-116 cells with IC50 values of 6.99 ± 0.5, 7.44 ± 0.4, and 8.15 ± 0.8 µM, respectively, and MCF-7 cells with IC50 values of 7.89 ± 0.7, 8.24 ± 0.7, and 9.32 ± 0.7 µM, respectively, in comparison with sorafenib as reference drug with IC50 values of 9.18 ± 0.6, 5.47 ± 0.3, and 7.26 ± 0.3 µM, respectively. The most active compounds 4a-c, 9b,c,e,f,h, and 11c,e were further evaluated for their VEGFR-2 inhibition. Compounds 4c and 4b potently inhibited VEGFR-2 at IC50 values of 0.12 ± 0.01 and 0.13 ± 0.02 µM, respectively, which are nearly equipotent to the sorafenib IC50 value (0.10 ± 0.02 µM). Furthermore, molecular docking studies were performed for all synthesized compounds to assess their binding pattern and affinity toward the VEGFR-2 active site.

Evaluation of drug candidature: In silico ADMET, binding interactions with CDK7 and normal cell line studies of potentially anti-breast cancer enamidines

We have recently explored novel class of potentially anti-breast cancer active enamidines in which four molecules 4a-c and 4h showed higher anticancer activity compared to standard drug doxorubicin. As a part of extension of this work, we have further evaluated in silico cheminformatic studies on bioactivity prediction of synthesized series of enamidines using mole information. The normal cell line study of four lead compounds 4a-c and 4h against African green monkey kidney vero strain further revealed that the compounds complemented good selectivity in inhibition of cancer cells. The in silico bioactivity and molecular docking studies also revealed that the compounds have significant interactions with the drug targets. The results reveal that enamidine moieties are vital for anti-breast cancer activity as they possess excellent drug-like characteristics, being potentially good inhibitors of cyclin dependent kinases7 (CDK7).

Synthesis, antibacterial evaluation, and DNA gyrase inhibition profile of some new quinoline hybrids.

Antibiotic-resistant bacteria continue to play an important role in human health and disease. Inventive strategies are necessary to develop new therapeutic leads to challenge drug-resistance problems. From this perception, new quinoline hybrids bearing bioactive pharmacophores were synthesized. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against nine bacterial pathogenic strains. The results revealed that most compounds exhibited good antibacterial activities. Seven compounds (2b, 3b, 4, 6, 8b, and 9c,d) displayed enhanced activity against methicillin-resistant Staphylococcus aureus compared to ampicillin. These compounds were subjected to an in vitro S. aureus DNA gyrase ATPase inhibition study, which revealed that compounds 8b, 9c, and 9d showed the highest inhibitory activity with IC50 values of 1.89, 2.73, and 2.14 μM, respectively, comparable to novobiocin (IC50 , 1.636 μM). Compounds 2a-c, 3a, 7c, 9c,d, and 10a,b revealed half the potency of levofloxacin in inhibiting the growth of Pseudomonas aeruginosa. As an attempt to rationalize the observed antibacterial activity for the most active compounds 8b, 9c, and 9d, molecular docking in the ATP binding site of S. aureus gyrase B was performed using Glide. Such compounds could be considered as promising scaffolds for the development of new potent antibacterial agents.

Strategy for Extending the Nitrogen Chain: The Bis(1,2,3-triazole) Formation Reaction from Tosylhydrazones and N-Amino Azole.

A facile and versatile synthesis strategy for the bis(1,2,3-triazole) formation reaction was developed from tosylhydrazones and N-amino (N-NH2) azole instead of C-amino amine derivatives. The novel energetic compounds containing the bicycle catenated six-nitrogen chain (N6) and the first example of N7 neutral compounds were synthesized in a moderate or high yield. The possible mechanism of bis(1,2,3-triazole) formation reaction based on amino azole of N-NH2 was verified by the X-ray crystal structure of key intermediates. In addition, four energetic compounds 4aa, 4ba, 4ac, and 4ad containing N6 and N7 structures possess acceptable decomposition temperatures (150.1-201.6 °C) and moderate calculated detonation performances (6850-7727 m/s). Among them, 4aa (N6 structure) and 4ad (N7 structure) could be used as the melt-cast explosive candidate and the gas generation agent candidate, respectively. This type of nitrogen-nitrogen bonding formation opens a new method for discovery of novel high-nitrogen energetic compounds containing catenated multiple nitrogen atoms especially odd number nitrogen compounds.

Design, synthesis of 4,5-diazafluorene derivatives and their anticancer activity via targeting telomeric DNA G-quadruplex

In our work, 19 novel 4,5-diazafluorene derivatives (11a-d, 12a-d, 13a-d, 14a-c, 15c, 16a-c) bearing a 1,3-disubstituted pyrazol/thioxothiazolidinone or thioxothiazolidinone-oxadiazole moieties were designed, synthesized, preliminarily explored for their antitumor activities and in vitro mechanism. All compounds showed different values of antiproliferative activity against A549, AGS, HepG2 and MCF-7 cell lines through CCK-8. Especially, the compound 14c exhibited the strongest activity and best selectivity against A549 cells with an IC50 1.13 μM and an SI value of 7.01 relative to MRC-5 cells, which was better than cisplatin (SI = 1.80) as a positive control. Experimental results at extracellular level demonstrated that compounds 14a-c could strongly interact with the G-quadruplex(es) formed in a 26 nt telomeric G-rich DNA, in particular, the 14c exhibits quite strong binding affinity with an association equilibrium constant (KA) of 7.04(±0.16) × 107 M−1 and more than 1000-fold specificity to G4-DNA over ds-DNA and Mut-DNA at the compound/G4-DNA ratio of 1:1. Further trap assay ascertained that compounds 14a-c owned strong inhibitory ability of telomerase activity in A549 cells, suggesting that these compounds have great possibility to target telomeric G-quadruplexes and consequently indirectly inhibit the telomerase activity. In addition, it is worthy of note that the remarkable inhibitory effects of 14a-c on the mobility of tested cancer cells were observed by wound healing assays. Furthermore, molecular docking and UV–Vis spectral results unclose the rationale for the interaction of compounds with such G-quadruplex(es). These results indicate that the growth and metastasis inhibition of cancer cells mediated by these 4,5-diazafluorene derivatives possibly result from their interaction with telomeric G-quadruplexes, suggesting that 4,5-diazafluorene derivatives, especially 14c, possess potential as anticancer drugs.

Pyrazole and Nicotinonitrile Derivatives Synthesized from Sulfa Drugs, and Their Antibacterial Activity

Utility of sulfadiazine and sulfaguanidine in synthesis of new series of pyrazoles and 2-pyridones is described. Diazotization of sulfadiazine that results in formation of diazonium salt 2 followed by coupling of the latter with active methylene compounds leads to hydrazinyl derivatives 3a–3d. Heterocyclization of compounds 3a–3c with hydrazine derivatives gives to a series of pyrazole derivatives 4–6. Similarly, a series of pyrazole derivatives 12–14 is developed from sulfaguanidine. Condensation of hydrazinyl derivatives 3a–3c with cyanoacetamide in the presence of a catalytic amount of piperidine gives 2-oxonicotinonitriles 15a–15c. Tests of the new synthesized compounds against four pathogenic gram (+ve) and gram (−ve) bacteria demonstrate their activity higher than that of the standard Amoxicillin.

Synthesis and DFT Quantum Chemical Calculations of 2-Oxopyrimidin-1(2H)-yl-Urea and Thiorea Derivatives

A series of the new 2-oxopyrimidin-1(2H)-yl-urea (3a-c) and thiourea (4a-d) derivatives were synthesized by the reaction of arylisocyanates (2a-c) or arylisothiocyanates (2d-g) and the 1-amino-5-(4-methoxybenzoyl)-4-(4-methoxyphenyl)pyrimidin-2(1H)-one (1). The structures of the compounds 3a-c and 4a-d were characterized by elemental analysis, FT-IR, 1H and 13C-NMR spectroscopic techniques. In addition to experimental study in order to find molecular properties, quantum-chemical calculations of the synthesized compounds were carried out by using DFT/B3LYP method with basis set of the 6-311G(d,p). Quantum chemical features such as HOMO, LUMO, HOMO-LUMO energy gap, Ionization potential, chemical hardness, chemical softness, electronegativity, chemical potential, dipole moment etc. values for gas and solvent phase of neutral molecules were calculated and discussed.

Novobiocin-ferrocene conjugates possessing anticancer and antiplasmodial activity independent of HSP90 inhibition.

A series of tailored novobiocin-ferrocene conjugates was prepared in moderate yields and investigated for in vitro anticancer and antiplasmodial activity against the MDA-MB-231 breast cancer line and Plasmodium falciparum 3D7 strain, respectively. While the target compounds displayed moderate anticancer activity against the breast cancer cell line with IC50 values in the mid-micromolar range, compounds 10a-c displayed promising antiplasmodial activity as low as 0.889µM. Furthermore, the most promising compounds were tested for inhibitory effects against a postulated target, heat shock protein 90 (Hsp90). A selection of tailored novobiocin derivatives bearing the organometallic ferrocene unit were synthesized and characterized by common spectroscopic techniques. The target compounds were investigated for in vitro anticancer and antimalarial activity against the MDA-MB-231 breast cancer cell line and Plasmodium falciparum 3D7 strain, respectively.

Effect of Ancillary Ligand in Cyclometalated Ru(II)-NHC-Catalyzed Transfer Hydrogenation of Unsaturated Compounds.

In an effort to develop efficient Ru(II)-NHC-based catalyst considering their stereoelectronic effect for hydride-transfer reaction, we found that the ancillary NHC ligand can play a significant role in its catalytic performance. This effect is demonstrated by comparing the activity of two different types of orthometalated precatalysts of general formula [( p-cymene)(NHC)RuII(X)] (NHC = an imidazolylidene-based ImNHC, compound 2a-c, or a mesoionic triazolylidene-based tzNHC, compound 4) in transfer hydrogenation of carbonyl substrates. The electron-rich precatalyst, 2c, containing p-OMe-substituted NHC ligand performed significantly better than both unsubstituted complex 2a and p-CF3 substituted electron-poor complex 2b in ketone reduction. Whereas bulky mesoionic triazolylidene ligand containing complex 4 was found to be superior catalyst for aldehyde reduction and the precatalyst 2a is more suitable for the selective transfer hydrogenation of a wide range of aromatic aldimines to amines. To the best of our knowledge, this is the first systematic study on the effect of stereoelectronic tuning of ancillary orthometalated NHC ligand in Ru(II)-catalyzed transfer hydrogenations of various types of unsaturated compounds with broad substrate scope.

Drug likeness, targets, molecular docking and ADMET studies for some indolizine derivatives.

The aim of this work was to investigate the biomolecular targets for a library of indolizines, study their molecular properties, drug likeness, target prediction, performing docking studies and exploring their ADMET profile in search for a lead compound. All compounds appeared to comply with Lipinski's rule without any violation, additionally their solubility is viewed good except for compounds 4a-c which are anticipated to be reasonably soluble, their Milog P score was 4.18-4.9, proposing that these compounds are the most lipophilic with least water solubility, however this may be helpful as the cannabinoid receptor-1 is the most probable target for these three compounds. The inclusive target for the selected library was tau protein. Structure based studies demonstrated great fitting of indolizines with tau protein, along these lines they are expected to have pharmacological action in vivo. This urged us to think about the ADMET properties of this library. These investigations suggested the ability of the selected compounds to pass the blood brain barrier (BBB) (aside from them compounds 2c and 3c) and affect tau proteins, which will be valuable for the treatment of Alzheimer's disease, particularly compound 5 which does not require any SAR modifications to attain the BBB.

A New Type of Three-Dimensional Hybrid Polymeric Haloplumbate Based on Rare High-Nuclear Heterometallic Clusters.

A series of new three-dimensional hybrid heterometallic haloplumbates [Pb8M(μ3-O)2X8(XH)(eg)3] n {H2eg = ethylene glycol; X = Cl, M = Co (1a), Ni (1b), Zn (1c); X = Br, M = Ni (2a), Zn (2b); X = I, M = Zn (3)} have been solvothermally synthesized and structurally characterized. Compounds 1a-c, 2a-b, and 3 consist of one-dimensional haloplumbate [Pb2X9] chains, and heptanuclear heterometallic [Pb6M(μ3-O)2(eg)3] clusters based on two [Pb3(μ3-O)(eg)1.5] cores share one M2+ ion, which are interconnected to generate three-dimensional heterometallic frameworks. Although a few heterometallic haloplumbates incorporating other metal complexes have been reported, they usually exhibit low-dimensional structures. The present heterometallic haloplumbates offer good examples of applying high-nuclear heterometallic [Pb6MO2(eg)3] clusters to construct a new type of high-dimensional hybrid heterometallic haloplumbate. Compounds 1a-b and 2a indicate unusual ferrimagnetic behavior. Their optical properties are investigated at room temperature, and density functional theory calculations of compounds 1c, 2b, and 3 are also studied.

Design, synthesis and identification of novel substituted isothiochromene analogs as potential antiviral and cytotoxic agents

In this study, we present the synthesis of novel isothiochromenes, thiazolidonone, thiazinone, arylidines, triazoles, and pyrimidinone compounds based on the starting material 3-amino- isothiochromene-4-carbonitrile 3. The chemical structures were confirmed using spectroscopic methods and elemental analyses. These compounds were screened for their in vitro antiviral and antitumor activities. Compounds 10a-c and 22a-b showed activity against herpes simplex virus-1 (HSV-1) and human immunodeficiency virus-1 (HIV-1). Compounds 15 and 21a-b exhibited activity against various types of cancer cell lines.

Trichlorostannyl complexes of Ruthenium(II): Synthesis, structure, reactivity and computational studies

Trichlorostannyl complexes [Ru(SnCl3) (Cp')L] (2a-c) were prepared by treatment of optically active half-sandwich chlorocomplexes [RuCl(Cp')L] (1a-c) with an excess of SnCl2.2H2O in ethanol. Treatment of trichlorostannyl complexes 2a-c with NaBH4 afforded trihydridostannyl derivatives [Ru(SnH3) (Cp')L] (3a-c) in moderated yields. Treatment of 2a-c with MgBrMe gave the trimethylstannyl complexes Ru(SnMe3) (Cp')L (4a-c). Alkynylstannyl derivatives [Ru{Sn(C≡CPh)3}(Cp')L] (5a-c) were prepared by treatment of trichlorostannyl compounds 2a-c with an excess of LiC≡CPh in thf. All the complexes present optical activity. The complexes were characterized spectroscopically and by X-ray crystal structure determination of [RuCl(η5-C5Me5)L] (1b), [Ru(SnCl3) (η5-C5Me5)L] (2b), and [Ru(SnCl3) (η5-C9H7)L] (2c). The influence of different ligands on the RuP interaction in several complexes 1a-c, 2a-c and 3a-c was evaluated by DFT calculations. These calculations indicate that [SnCl3]- has a stronger stabilization effect than [Cl]- and the same occurs between C9H7 and C5Me5. These relative stabilities combined with the distortion energies of the fragments produce a stabilizing effect in the RuP bonds of complex 2c that is twice as strong as in the 1b complex.

Synthesis of Novel Thieno[2,3-d]pyrimidine Derivatives and Evaluation of Their Cytotoxicity and EGFR Inhibitory Activity

4-Substitutedaminoquinazoline scaffolds were reported to possess potent cytotoxic and EGFR inhibitory activity such as gefitinib (Iressa), erlotinib (Tarceva) and tandutinib. Synthesis of novel 4-substitutedaminothieno[2,3-d]pyrimidine derivatives as bioisosters of 4-substitutedaminoquinazoline derivatives with potential cytotoxic and EGFR inhibitory activity. Novel 4-substitutedaminothieno[2,3-d]pyrimidine derivatives 4a-i and 5a-c were synthesized via reacting corresponding 4-chlorothieno[2,3-d]pyrimidine derivatives 3a-c with N-methylpiperazine, morpholine, N-phenylpiperazine or 1,3-propanediamine. Six compounds (2a, 4d, 4e, 5a-c) were selected by the National Cancer Institute (USA) for evaluating their cytotoxic activity using 60 different human tumor cell lines using a single dose (10-5 Molar). The rest of the synthesized compounds (2b, 2c, 3a-c, 4a-c and 4f-i) were subjected to screening against T47D breast cancer cell line using a single dose (10-5 Molar) at Pharmacology lab., Cancer biology lab., Egyptian National Institute. Moreover, compounds 2a and 4b-e were subjected to further evaluation by IC50 determination. Finally, the inhibition of epidermal growth factor receptor (EGFR) was then investigated for the most active compounds 2a and 4d. Compounds 2a and 4b-e showed significant cytotoxic activity. Compound 2a was more potent than doxorubicin against lung cancer cell line A549 with IC50 = 13.40 μM and comparable activity against MCF7. Compound 4d exhibited more potent activity than Doxorubicin against prostate PC3 (IC50 = 14.13 µM) while showed comparable activity against MCF7 and T47D. 4-Substitutedaminothieno[2,3-d]pyrimidine is a promising backbone for the design and synthesis of potent cytotoxic leads.

Structure-based design of some isonicotinic acid hydrazide analogues as potential antitubercular agents

New pyridine derivatives were designed and synthesized as Isonicotinic acid hydrazide (INH) analogues. The synthesized compounds were evaluated for their antitubercular activity against Mycobacterium tuberculosis strain H37Rv. Ten compounds (3c, 3e-g, 5a-c, 6h, 10 and 11b) showed promising antitubercular activity with MIC range 7.30 µM–19.39 µM. Compounds 3e, 3g, 5b and 11b were the most potent analogues, with MIC 7.30–8.74 µM. They were equipotent to the standard drug Ethambutol (MIC 7.64 µM) and more active than the standard drug Pyrazinamide (MIC 50.77 µM). They were further examined for cytotoxicity in human embryonic kidney (HEK) cell line at the concentration of 50 µg/mL using MTT assay. Results declared that most compounds showed acceptable safety margin. Molecular Docking studies into 2-trans-enoyl-acyl carrier protein reductase, called InhA have been conducted for compounds 3e, 3g, 5b and 11b using Molecular Operating Enviroment software (MOE 2016.0802), where reasonable binding interactions have been identified and effective overall docking scores have been recorded. Their drug-likeness has been assessed using Molinspiration and Osiris software.

Synthesis and antitumor evaluation of novel 4-anilino-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate derivatives as potential EGFR inhibitors.

Novel series of 4-anilino-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylates (5a-p, 7, and 8a-e) were synthesized and evaluated for their antiproliferative activity against the A549, HT29, H460, and H1975 cancer cell lines in vitro. Nine compounds (5a-c, 5i, 5j, 7, 8a, 8b, 8i) demonstrated moderate to significant cytotoxic activity with IC50 values below 18 µM on A549 cells, which were comparable to that of the reference gefitinib (IC50 = 18.44 µM). Especially, the further enzymatic analysis on epidermal growth factor receptor (EGFR), HER2, and VEGFR identified compound 5a as a promising hit that exhibited considerable potency both in cellular (IC50 = 5.67, 17.04, 11.29, and 12.65 µM, respectively) and EGFR enzymatic assays (IC50 = 14.8 nM). Compound 5a was capable of down-regulating the expression of EGFR and inhibited EGFR phosphorylation in a dose-dependent manner, representing a potential EGFR candidate for further optimization.

Synthesis and biological evaluation of new coumarins bearing 2,4-diaminothiazole-5-carbonyl moiety

A series of new coumarin-containing compounds 3a-l and 4a-c was designed and synthesized based on the chalcone-type 4-amino-5-cinnamoylthiazole scaffold 2, and screened for their in vitro anticancer and antioxidant activities. Representatively, the 2-thiomorpholinothiazole derivative 3k with IC50 values of 7.5–16.9 μg/ml demonstrated good cytotoxic effects against tested cell lines MCF-7, HepG2 and SW480. Further investigation by flow cytometric analysis confirmed that this compound induces apoptotic cell death in MCF-7 cells and cause G1-phase arrest in the cell cycle. Moreover, most of compounds had intrinsic potential for radical scavenging activity and ferric-reducing power as investigated by DPPH and FRAP assays.