Biopharmaceutical studies of 3-substituted isatin derivatives

The metabolic fate of isatin hydrazone (Ia), isatin-3-thiosemicarbazone (Ib), isatin-3-semicarbazone (Ic), isatin-3-phenylhydrazone (Id), isatin oxime (Ie) and 3-hydroxy-3-acetonyl oxindole (II) was studied in rabbits. The compounds were administered orally in the dose of 300 mg/kg body wt. Isatin anthranilic acid, tryptophan and nicotinic acid were identified as the major metabolites excreted in urine. The 3-hydroxy-3-acetonyl oxindole (II) gave on additional metabolite, oxindole. The major metabolites were separated and identified unambiguously on thin layer silica gel plate. Metabolic pathways have been proposed to explain the biotransformation of the compounds investigated.     

Synthesis of 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and their effects on neuronal cell death

Novel 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole analogs were synthesized by either of the following two pathways: (1) a sequence of Knoevenagel condensation of oxindole with (4-dimethylamino)cinnamaldehyde–hydrogenation, or (2) alkylation of oxindole dianion with [(4-dimethylamino)phenyl]alkyl halides. Subsequent alkylation at C-3 and/or N-1 of the oxindole skeleton by anion-based methods provided additional substituted derivatives for structure-activity relationship studies. Their effects on neuronal cell death induced by oxidative stress were evaluated by lactate dehydrogenase assay. Compounds with the alkyl chain length of 2–4 significantly suppressed the neuronal cell death. No significant change occurred in the activity by substitution with less-polar groups. The stereochemistry at C-3 of the oxindole core was also irrelevant for the neuroprotective effects of these compounds.     

Auxin activity of 3-hydroxymethyl oxindole and 3-methylene oxindole in oat

Two photooxidation products of indol-3-ylacetic acid (IAA), 3-hydroxymethyl oxindole (HMO) and 3-methylene oxindole (Meox) were almost as effective as IAA in stimulation of growth of oat (Avena saliva cv. Kent) coleoptile sections. The IAA failed to give stimulation of growth after pretreatment of the coleoptiles with chemicals inhibiting oxidation of IAA, but the growth promoting effects of HMO or Meox remained unaffected by such pretreatment. After pretreatment with the chemicals which can form an adduct with Meox, the growth promoting activities of IAA, HMO or Meox were lost showing the involvement of the oxindole pathway of IAA metabolism in IAA action. Time-course experiments also showed that the stimulatory effects of HMO and Meox were the same as IAA.     

Novel 3,3-disubstituted oxindole derivatives. Synthesis and evaluation of the anti-proliferative activity

3,3-Disubstituted oxindole derivatives bearing a nitrogen atom at the C-3 position have been synthesized starting from 3-alkyl oxindole through a metal free pathway. These derivatives have been tested in five human tumor cell lines (PC3, MCF7, SW620, MiaPaca2 and A375) and on primary cells (PBMCs) from healthy donors providing compound 6d showing a strong anticancer effect in all cancer lines on the low micromolar range.     

Environmental factors affecting indole metabolism under anaerobic conditions

The influence of physiological and environmental factors on the accumulation of oxindole during anaerobic indole metabolism was investigated by high-performance liquid chromatography. Under methanogenic conditions, indole was temporarily converted to oxindole in stoichiometric amounts in media inoculated with three freshwater sediments and an organic soil. In media inoculated with methanogenic sewage sludge, the modest amounts of oxindole detected at 35 degrees C reached higher concentrations and persisted longer when the incubation temperature was decreased from 35 to 15 degrees C. Also, decreasing the concentration of sewage sludge used as an inoculum from 50 to 1% caused an increase in the accumulation of oxindole from 10 to 75% of the indole added. Under denitrifying conditions, regardless of the concentration or source of the inoculum, oxindole appeared in trace amounts but did not accumulate during indole metabolism. In addition, denitrifying consortia which previously metabolized indole degraded oxindole with no lag period. Our data suggest that oxindole accumulation under methanogenic, but not under denitrifying conditions is caused by differences between relative rates of oxindole production and destruction.     

Environmental factors affecting indole metabolism under anaerobic conditions.

The influence of physiological and environmental factors on the accumulation of oxindole during anaerobic indole metabolism was investigated by high-performance liquid chromatography. Under methanogenic conditions, indole was temporarily converted to oxindole in stoichiometric amounts in media inoculated with three freshwater sediments and an organic soil. In media inoculated with methanogenic sewage sludge, the modest amounts of oxindole detected at 35 degrees C reached higher concentrations and persisted longer when the incubation temperature was decreased from 35 to 15 degrees C. Also, decreasing the concentration of sewage sludge used as an inoculum from 50 to 1% caused an increase in the accumulation of oxindole from 10 to 75% of the indole added. Under denitrifying conditions, regardless of the concentration or source of the inoculum, oxindole appeared in trace amounts but did not accumulate during indole metabolism. In addition, denitrifying consortia which previously metabolized indole degraded oxindole with no lag period. Our data suggest that oxindole accumulation under methanogenic, but not under denitrifying conditions is caused by differences between relative rates of oxindole production and destruction.     

Structure-activity relationships of the oxindole growth hormone secretagogues

A series of substituted oxindole derivatives of SM-130686 was synthesized and evaluated as ghrelin receptor agonists. Modification of the substituents on the C3-aromatic part of the oxindole led to compounds with subnanomolar binding affinities. Compound 4i (IC(50)=0.02 nM) was orally active at low doses and showed in vivo activity when orally administered, 2 mg/kg twice a day for 4 days, as evidenced by significant body weight gain.     

Investigation of Uña De Gato I. 7-Deoxyloganic acid and 15N NMR spectroscopic studies on pentacyclic oxindole alkaloids from Uncaria tomentosa

The C-8-(S) isomer of deoxyloganic acid (7-deoxyloganic acid), together with beta-sitosteryl glucoside, five known stereoisomeric pentacyclic oxindole alkaloids and the tetracyclic oxindole isorhyncophylline, were isolated from the inner bark of Uncaria tomentosa. Structures of the isolated compounds were based on 1H and 13C NMR data, mainly 2D NMR experiments, including 1H-13C HMBC and 1H-1H NOESY correlation. Furthermore, the hitherto unreported 15N chemical shifts of the isomeric oxindole alkaloids, using 1H-15N HMBC experiments, were utilized to facilitate their characterization. Uncarine D showed weak cytotoxic activity against SK-MEL, KB, BT-549 and SK-OV-3 cell lines with IC(50) values between 30 and 40 microg/ml, while uncarine C exhibited weak cytotoxicity only against ovarian carcinoma (IC(50) at 37 microg/ml).     

Oxindole, a Sedative Tryptophan Metabolite, Accumulates in Blood and Brain of Rats with Acute Hepatic Failure

Abstract: Rats treated with oxindole (10–100 mg/kg i.p.), a putative tryptophan metabolite, showed decreased spontaneous locomotor activity, loss of the righting reflex, hypotension, and reversible coma. Brain oxindole levels were 0.05 ± 0.01 nmol/g in controls and increased to 8.1 ± 1.7 or 103 ± 15 nmol/g after its administration at doses of 10 or 100 mg/kg i.p., respectively. To study the role that oxindole plays in the neurological symptoms associated with acute liver failure, we measured the changes of its concentration in the brain after massive liver damage, and we investigated the possible metabolic pathways leading to its synthesis. Rats treated with either thioacetamide (0.2 and 0.4 g/kg i.p., twice) or galactosamine (1 and 2 g/kg i.p.) showed acute liver failure and a large increase in blood or brain oxindole concentrations (from 0.05 ± 0.01 nmol/g in brains of controls to 1.8 ± 0.3 nmol/g in brains of thioacetamide-treated animals). Administration of tryptophan (300–1,000 mg/kg p.o.) caused a twofold increase, whereas administration of indole (10–100 mg/kg p.o.) caused a 200-fold increase, of oxindole content in liver, blood, and brain, thus suggesting that indole formation from tryptophan is a limiting step in oxindole synthesis. Oral administration of neomycin, a broad-spectrum, locally acting antibiotic agent able to reduce intestinal flora, significantly decreased brain oxindole content. Taken together, our data show that oxindole is a neurodepressant tryptophan metabolite and suggest that it may play a significant role in the neurological symptoms associated with acute liver impairment.     

Four tetracyclic oxindole alkaloids and a taberpsychine derivative from a Malayan Tabernaemontana

Four tetracyclic oxindole alkaloids, 7(R)- and 7(S)-geissoschizol oxindole (1 and 2), 7(R),16(R)- and 7(S),16(R)–19(E)-isositsirikine oxindole (3 and 4), in addition to a taberpsychine derivative, N(4)-demethyltaberpsychine (5), were isolated from the Malayan Tabernaemontana corymbosa and the structures were established using NMR and MS analysis.     

Direct Asymmetric anti‐Mannich‐Type Reactions Catalyzed by Cinchona Alkaloid Derivatives

A series of cinchona alkaloid derivatives were used to catalyze the asymmetric anti‐Mannich‐type reaction of 3‐methyl‐2‐oxindole with N‐tosyl aryl aldimines. The resulting anti‐3,3‐disubstituted 2‐oxindole products were obtained in good yields (up to 92%) with high diastereo‐ and enantioselectivities (anti/syn up to 97:3 and 91% ee). Chirality 26:801–805, 2014. © 2014 Wiley Periodicals, Inc.     

Nb-demethylalstophylline oxindole an oxindole alkaloid from the leaves of Alstonia macrophylla

An oxindole alkaloid, N_b-demethylalstophylline oxindole has been isolated from the leaves of Alstonia macrophylla. Its structure has been elucidated on the basis of spectroscopic studies.     

Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.     

Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.     

Heat-Melt of β-1, 3-d-Glucan Gel

Six new products of oxidation of indolyl-3-acetic add catalyzed by horseradish peroxidase were isolated, along with four known ones, 3-hydroxymethyloxindole (1), 3-methyleneoxindole (2), indolyl-3-aldehyde (4), and 3,3-diindolylmethane (10). Based on spectroscopic and chemical evidence, the new products were identified as 3-acetoxyindole (3), 3-(indol-3-ylmethyl)oxindole (6), 3-[(2-mdol-3-ylmethyl)indol-3-ylmethyl]oxindole (9), the 3-hydroxymethyl compounds of 6 and 9 (5 and 7), and 2-(indol-3-ylmethyl)indolyl-3-acetic acid (8), respectively.     

Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst

Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.     

Novel oxindole/benzofuran hybrids as potential dual CDK2/GSK-3β inhibitors targeting breast cancer: design,synthesis, biological evaluation,and in silico studies

The serine/threonine protein kinases CDK2 and GSK-3β are key oncotargets in breast cancer cell lines, therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised as dual CDK2/GSK-3β inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N¹-unsubstituted oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast cancer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC₅₀ of 3.41, 3.45 and 2.27 μM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 μM, respectively, on T-47D cell lines, in comparison to the used reference standard (staurosporine) IC₅₀ of 4.81 and 4.34 μM, respectively. On the other hand, the N¹-substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activity on both breast cancer cell lines. CDK2 and GSK-3β enzyme inhibition assay of series 5 revealed that compounds 5d and 5f are showing potent dual CDK2/GSK-3β inhibitory activity with IC₅₀ of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3β. The most potent compounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of the CDK2/GSK-3β inhibition. Molecular docking studies showed that the newly synthesised N¹-unsubstituted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3β. The oxindole ring is accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in GSK-3β. Whereas, in series 7 and 13, the N¹-substitutions on the oxindole nucleus hinder the compounds from achieving these key interactions with hinge region amino acids what rationalises their moderate to low anti-proliferative activity.     

SAR studies of 6-aryl-1,3-dihydrobenzimidazol-2-ones as progesterone receptor antagonists

We have previously reported that the aryl substituted benzimidazolones, benzoxazinones, and oxindoles (e.g., 1-3) are progesterone receptor (PR) antagonists and have recently disclosed that the nature of 5- and 6-aryl moieties played a critical role in PR functional activity in the oxindole and benzoxazinone templates. For example, replacing the phenyl group of PR antagonists 2 and 3 with a 5'-cyanopyrrol-2'-yl moiety switched their functional activity to PR agonist activity (2a and 3a). These findings prompted us to examine if there is a similar effect of the 6-aryl moieties on the PR functional activity for the benzimidazolone template. Numerous analogs, such as 5, showed potent PR antagonist activity with about a 10-fold increase in potency as compared to those reported earlier in the same series. More interestingly, pyrrole-containing benzimidazolones 24-27 remained as PR antagonists in contrast to the PR agonist activity switch for oxindole and benzoxazinone scaffolds when a 5'-cyanopyrrol-2'-yl group was installed as a pendant aryl group.     

Discovery of XEN907, a spirooxindole blocker of NaV1.7 for the treatment of pain

Starting from the oxindole 2a identified through a high-throughput screening campaign, a series of Na_V1.7 blockers were developed. Following the elimination of undesirable structural features, preliminary optimization of the oxindole C-3 and N-1 substituents afforded the simplified analogue 9b, which demonstrated a 10-fold increase in target potency versus the original HTS hit. A scaffold rigidification strategy then led to the discovery of XEN907, a novel spirooxindole Na_V1.7 blocker. This lead compound, which in turn showed a further 10-fold increase in potency, represents a promising structure for further optimization efforts.     

Raman spectra of chemically modified lysozyme. Oxindole derivatives.

The Raman spectra of oxidation products of lysozyme have been investigated. The protein was oxidized by N-bromosuccinimide and dimethyl sulfoxide/HCl. Depending on the experimental conditions one to six tryptophan residues are oxidized to oxindole. The most prominent difference between the spectra of lysozyme and its oxindole derivatives is the strong band at 1017 cm^?1 which displaces the tryptophan peak at 1010 cm^?1. Other tryptophan bands are also weakened corresponding to the number of the tryptophan side chains destroyed. Shifts are observed in the amide I and in the amide III regions sensitive to conformational changes. These shifts indicate conformational differences in the higher oxidized species and in the native enzyme, although the amide III maxima overlap with a strong oxindole band. Similar effects are observed in the range of the C-C stretching vibrations of the peptide backbone. If more than one tryptophan side chain is oxidized changes have also been found in the S-S stretching range. The evaluation of this effect is difficult because of the strong oxindole vibration appearing in this region. In species oxidized by great excess of N-bromosuccinimide the tyrosine vibrations can no longer be detected, indicating the modification of this amino acid too.