Microwave-Assisted Synthesis of 3-Hydroxy-2-oxindoles and Pilot Evaluation of Their Antiglaucomic Activity

Glaucoma is a widespread neurodegenerative disease for which increased intraocular pressure (IOP) is a primary modifiable risk factor. Recently, we have observed that compounds with oxindole scaffolds are involved in the regulation of intraocular pressure and therefore have potential antiglaucomic activity. In this article, we present an efficient method for obtaining novel 2-oxindole derivatives via microwave-assisted (MW) decarboxylative condensation of substituted isatins with malonic and cyanoacetic acids. Various 3-hydroxy-2-oxindoles were synthesized using MW activation for 5–10 min with high yields (up to 98%). The influence of novel compounds applied in instillations on IOP was studied in vivo on normotensive rabbits. The lead compound was found to reduce the IOP by 5.6 Torr (ΔIOP for the widely used antiglaucomatousic drug timolol 3.5 Torr and for melatonin 2.7 Torr).


Introduction
Glaucoma is the most frequent cause of irreversible blindness since it leads to the irreversible loss of retinal ganglion cells and optic nerve fibers [1][2][3].Unfortunately, at the moment there are few effective pharmacological ways of treating glaucomatous optic neuropathy.The conventional, important, and commonly used way of therapy is a life-long daily use of hypotensive drugs to slow down the progression of optic nerve damage [4,5].Surgical methods also do not provide a total cure.The therapeutic effect of surgery lasts from one to five years, depending on the type of glaucoma (open-angle or angle-closure) and the stage of the disease [6].
As a rule, the primary treatment of open-angle glaucoma is carried out through pharmacotherapy.The main risk factor for glaucoma is ocular hypertension, which is an increase in intraocular pressure (IOP).The increase in IOP leads to damage to the optic nerve head, ischemia, and the loss of ganglion cells in the retina.Therefore, most antiglaucomic drugs are aimed at lowering IOP.Currently, there are six classes of ophthalmic drugs used in the treatment of glaucoma: prostaglandin analogues [7], carbonic anhydrase inhibitors [8], beta-blockers [9], alpha-adrenergic agonists [10], miotics [11], and rho-kinase (ROCK) inhibitors [12].The decrease in IOP occurs either due to a reduction in the production of aqueous humor by the ciliary body or due to an increase in uveoscleral outflow.Beta-blockers and carbonic anhydrase inhibitors work by the first mechanism, while prostaglandin analogs, miotics, and ROCK inhibitors work by the second.Alphaadrenergic agonists reduce the secretion of aqueous humor and at the same time increase the outflow [13].
A limited number of IOP-influencing molecular targets leads to the fact that patients develop tolerance and drugs no longer work.For example, some beta-blockers have a 30% chance of tachyphylaxis or drug tolerance development in 1 month [14][15][16].For other antiglautomatous drugs-up to 2-3 years for each new molecular target.Thus, the search for new compounds capable of lowering IOP to a safe level is an important and urgent task.
Recently, considerable attention has been paid to melatonin and dopamine receptors as promising targets in the search for antiglaucoma drugs [17,18], which causes interest in oxindole derivatives as well.Previously, we have observed that 2-oxindole derivatives can reduce IOP in animal models and have antioxidant properties [19][20][21][22][23].The most promising were recognized as 3-hydroxy-2-oxindoles.The standard method for obtaining such compounds is the reaction of aldol condensation of the starting isatins with carbonyl and carboxyl compounds.
In general, the synthesis of several 3-cyanomethyl-and 3-carboxymethyl-3-hydroxyoxindoles is carried out by the condensation of isatins with cyanoacetic or malonic acids with simultaneous decarboxylation [24].However, the long reaction time (3-72 h) prompted the search for a new efficient method for the synthesis of 3-hydroxy-2-oxindoles, containing cyano and carboxy groups in the side chain.For this purpose, the acceleration of the condensation of isatins with malonic acid derivatives under microwave irradiation was studied.
Condensation of isatins (1a-h) with cyanoacetic or malonic acids in the presence of an organic base under conventional reflux for 3 h leads to the formation of 3-hydroxy-3cyanomethyloxindoles (2a-h) and 3-hydroxy-3-carboxymethyloxindoles (3a-h), respectively (Scheme 1 and Table 1).We proposed that microwave irradiation can sufficiently increase the reaction rate.
tiglautomatous drugs-up to 2-3 years for each new molecular target.Thus, the search for new compounds capable of lowering IOP to a safe level is an important and urgent task.
Recently, considerable attention has been paid to melatonin and dopamine receptors as promising targets in the search for antiglaucoma drugs [17,18], which causes interest in oxindole derivatives as well.Previously, we have observed that 2-oxindole derivatives can reduce IOP in animal models and have antioxidant properties [19][20][21][22][23].The most promising were recognized as 3-hydroxy-2-oxindoles.The standard method for obtaining such compounds is the reaction of aldol condensation of the starting isatins with carbonyl and carboxyl compounds.
In general, the synthesis of several 3-cyanomethyl-and 3-carboxymethyl-3-hydroxyoxindoles is carried out by the condensation of isatins with cyanoacetic or malonic acids with simultaneous decarboxylation [24].However, the long reaction time (3-72 h) prompted the search for a new efficient method for the synthesis of 3-hydroxy-2-oxindoles, containing cyano and carboxy groups in the side chain.For this purpose, the acceleration of the condensation of isatins with malonic acid derivatives under microwave irradiation was studied.
Condensation of isatins (1a-h) with cyanoacetic or malonic acids in the presence of an organic base under conventional reflux for 3 h leads to the formation of 3-hydroxy-3cyanomethyloxindoles (2a-h) and 3-hydroxy-3-carboxymethyloxindoles (3a-h), respectively (Scheme 1 and Table 1).We proposed that microwave irradiation can sufficiently increase the reaction rate.

Thermal activation
Thermal activation -Et The optimization of MW reaction conditions was performed using 5-nitroisatin 1a and malonic acid (Table 2).The usage of other organic solvents, such as THF and EtOH, dramatically decreases the product yield.The piperidine and triethylamine were equally effective as base catalysts in this reaction, and the best results were obtained with 1.5 eq. of base in the case of malonic acid (see Table 2) and with 0.25 eq. of base for cyanoacetic acid.
The condensation of isatins (1a-h) with cyanoacetic and malonic acids proceeds with simultaneous decarboxylation by two alternative mechanisms, A and B. In the case of condensation with cyanoacetic acid, mechanism A is assumed (Scheme 2), while condensation with malonic acid probably proceeds by mechanism B (Scheme 3).Mechanism A involves first decarboxylation and the formation of acetonitrile enolate in situ, analogously to the condensation of aromatic beta-keto acids with isatins [25]. 1 Incomplete conversion of starting isatin.
The condensation of isatins (1a-h) with cyanoacetic and malonic acids proceeds with simultaneous decarboxylation by two alternative mechanisms, A and B. In the case of condensation with cyanoacetic acid, mechanism A is assumed (Scheme 2), while condensation with malonic acid probably proceeds by mechanism B (Scheme 3).Mechanism A involves first decarboxylation and the formation of acetonitrile enolate in situ, analogously to the condensation of aromatic beta-keto acids with isatins [25].Mechanism B involves first the nucleophilic addition of malonic acid to isatin followed by decarboxylation [26].This reaction mechanism indirectly confirms the fact that if the intensity of microwave irradiation is reduced, the product of the reaction of unsubstituted isatin 1g with malonic acid will not be 2-(3-hydroxy-2-oxoindolin-3-yl)acetic acid (3g), but 2-(3 -hydroxy-2-oxoindolin-3-yl)propanedioic acid (X) (scheme 3).
Usually, for compounds 2 and 3, the diastereotopically different CH2 protons are well resolved in 1H NMR spectra (see 2a, Figure 1).Interestingly, in the case of 7-nitro or 4nitrosubstituted cyanooxindoles, the diastereotopy in NMR was poorly resolved up to complete degradation into singlet for compounds 2b,d (Figure 1).The influence of all synthesized compounds 2 and 3 on IOP was tested in vivo using normotensive rabbits.According to the previous study of the dose-dependent influence Mechanism B involves first the nucleophilic addition of malonic acid to isatin followed by decarboxylation [26].This reaction mechanism indirectly confirms the fact that if the intensity of microwave irradiation is reduced, the product of the reaction of unsubstituted isatin 1g with malonic acid will not be 2-(3-hydroxy-2-oxoindolin-3-yl)acetic acid (3g), but 2-(3 -hydroxy-2-oxoindolin-3-yl)propanedioic acid (X) (scheme 3).
Usually, for compounds 2 and 3, the diastereotopically different CH2 protons are well resolved in 1H NMR spectra (see 2a, Figure 1).Interestingly, in the case of 7-nitro or 4nitrosubstituted cyanooxindoles, the diastereotopy in NMR was poorly resolved up to complete degradation into singlet for compounds 2b,d (Figure 1).The influence of all synthesized compounds 2 and 3 on IOP was tested in vivo using normotensive rabbits.According to the previous study of the dose-dependent influence Scheme 3. Proposed mechanism B for condensation of isatins with malonic acid.
Usually, for compounds 2 and 3, the diastereotopically different CH2 protons are well resolved in 1H NMR spectra (see 2a, Figure 1).Interestingly, in the case of 7-nitro or 4nitrosubstituted cyanooxindoles, the diastereotopy in NMR was poorly resolved up to complete degradation into singlet for compounds 2b,d (Figure 1).The influence of all synthesized compounds 2 and 3 on IOP was tested in vivo using normotensive rabbits.According to the previous study of the dose-dependent influence The influence of all synthesized compounds 2 and 3 on IOP was tested in vivo using normotensive rabbits.According to the previous study of the dose-dependent influence of melatonin and 2-oxindole derivatives on IOP [19], the instillation of 50 µL of 0.1%wt.solution was chosen for the pilot study of the new compounds' influence on IOP.
The obtained data are summarized in Table 3 and Figures 2 and 3. of melatonin and 2-oxindole derivatives on IOP [19], the instillation of 50 µ L of 0.1%wt.solution was chosen for the pilot study of the new compounds' influence on IOP.The obtained data are summarized in Table 3 and Figures 2 and 3.As can be seen in Table 3 and Figure 2, nitrile derivatives 2 are more active than acids 3, which have the same substituents in the 2-oxindole ring.5-Methoxy-2-oxindole derivatives showed a pronounced hypotensive effect, while the presence of nitro-groups decreased the hypotensive properties of compounds.The N-unsubstituted and N-benzylated compounds both retain the hypotensive activity, but the most active nitrile 2i (Figure 2) has both 5-OMe and N-benzyl groups.In comparison with oxindoles from our previous work [19], the advantages of the new compounds are their simpler synthesis and better water solubility, with a comparable hypotensive effect (∆IOP up to ca. 5 Torr).Compounds 2h and 2c, like timolol and melatonin, showed the maximum effect at 3 h and compound 2i-4 h after instillation, and all of them represent a more extended hypotensive effect than timolol and melatonin.The more significant hypotensive effects of these compounds (2c, 2h, and 2i) compared to melatonin or timolol 5 h after instillation are statistically significant (Figure 3).
All compounds were well tolerated by the animals.Visual examination revealed no such clinical signs of ocular irritation as conjunctival hyperemia, conjunctival or palpebral edema, lacrimation, or any reactions to discomfort (eye rubbing, avoidance, photophobia).

Chemistry
The 1,4-dioxane was distilled over sodium.The triethylamine and piperidine were dried over NaOH.Reactions were monitored by thin-layer chromatography (TLC) carried out on Merck TLC silica gel plates (60 F254), using UV light for visualization and basic aqueous potassium permanganate or iodine fumes as developing agents.Flash column chromatography purification was performed using silica gel 60 (particle size 0.040-0.060mm).The melting points were measured in open capillaries and are presented without correction. 1H and 13C NMR spectra were recorded at 298 K on a Bruker Avance 400 spectrometer with an operating frequency of 400 and 100 MHz, respectively, and calibrated using residual CHCl 3 (δH = 7.26 ppm) and CDCl 3 (δC = 77.16ppm) or DMSOd 5 (δH = 2.50 ppm) and DMSO-d 6 (δC = 39.52 ppm) as internal references.NMR data are presented as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet, br.= broad), coupling constant (J) in Hertz (Hz), integration.IR spectra were recorded on the Thermo Nicolet IR-200 in KBr or ZnSe.High-resolution mass spectra (HRMS) were measured on a Thermo Scientific LTQ Orbitrap instrument using nanoelectrospray ionization (nano-ESI).HPLC was measured using acetonitrile as the eluent on a Shimadzu Prominence LCMS-2020.
The reactions under microwave irradiation were carried out in a Monowave 300 microwave reactor (Anton Paar, GmbH), as well as in a Bosch HMT72M420 domestic microwave oven with a volume of 17 L. Reactions under thermal activation conditions were carried out on laboratory hot plates, as well as in a ChemiStation chemical reactor (EYELA).
All obtained compounds were sufficiently pure (89-98% based on HPLC and NMR, Files S1-S34) and could be used in biological activity evaluation and further synthesis without additional purification.
Method A: The reaction vessel was placed in a domestic microwave oven under radiation of 360W for 2 min.After that, the reaction vessel was slowly adjusted to ~50 • C, then quickly cooled to room temperature using a cool water bath and again placed in a domestic microwave oven for 2 min at the same power.The cycle of cooling and irradiation was repeated twice more.
Method B: The mixture in the reaction vessel was stirred with reflux for 3 h.Method C: The reaction vessel was placed in a microwave reactor.In the device settings, the temperature was set to 120 • C, the stirring intensity to 600 rpm, and the time to 3 min.Every 3 min, the reaction flask was cooled to room temperature.The cycle of cooling and irradiation was repeated twice more.
The mixture in the reaction vessel was stirred with reflux for 3 h.Purification (common for all above methods): The reaction mixture was evaporated to dry in vacuo.The residue was washed with 3 mL of 10% HCl.The product was dissolved in 5 mL EtOAc, dried over Na 2 SO 4 (anhydrous), and the organic solvent was removed in vacuo.The following compounds had been obtained according to these procedures: (3-Hydroxy-5-nitro-2-oxo-2,3-dihydro-1H-indol-3-yl)acetonitrile (2a) [19].
NMR 13  3.1.2.Procedure for Synthesis of (3-Hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)malonic Acid (X) [26] A chemical test tube with a screw cap containing a reaction mixture was placed in a Bosch HMT72M420 domestic microwave oven (17 L volume) under radiation of 360W for 2 min.After that, the reaction vessel was cooled to room temperature.Briefly, 129 mg (0.88 mmol) of 1H-indole-2,3-dione (1g), 0.3 g (2.88 mmol) of malonic acid, 3 mL of pure dioxane, and 0.5 mL of triethylamine were involved in the reaction.A chemical test tube with a screw cap containing a reaction mixture was placed in a Bosch HMT72M420 domestic microwave oven (17 L volume) under radiation of 360W for 2 min.After that, the reaction vessel was cooled to room temperature.The reaction mixture was poured hot into a singlenecked round-bottom flask, and the organic solvent was removed in vacuo.The resulting compound was washed with small portions of 10% HCl.The product was dissolved in 5 mL EtOAc, dried over Na 2 SO 4 (anhydrous), and the organic solvent was removed in vacuo.One hundred and sixty-six milligrams of solid product was obtained.Yield 75%.

In Vivo Testing
The ability of oxindoles to reduce IOP was assessed on normotensive male Chinchilla rabbits weighing about 2 kg.All investigated substances were used topically as 0.1% solutions (w/v); in the case of melatonin, this corresponded to a concentration of 4.3 µM.All substances were dissolved in a 0.05 M phosphate buffer solution, pH 7.4, containing 5% dimethyl sulfoxide (DMSO) v/v.All compounds were initially dissolved in DMSO and then diluted to the required concentration with phosphate buffer solution.The solutions were instilled into both eyes at a fixed volume of 50 µL.IOP was measured before instillation and

Figure 1 .
Figure 1.Comparison of the 1H NMR characteristic signals of compounds 2a,b,d,f,e.

Figure 1 .
Figure 1.Comparison of the 1H NMR characteristic signals of compounds 2a,b,d,f,e.

Scheme 3 .
Scheme 3. Proposed mechanism B for condensation of isatins with malonic acid.

Figure 1 .
Figure 1.Comparison of the 1H NMR characteristic signals of compounds 2a,b,d,f,e.

Figure 1 .
Figure 1.Comparison of the 1H NMR characteristic signals of compounds 2a,b,d,f,e.

Figure 2 .Figure 3 .
Figure 2. Graphical representation of IOP reduction after instillation of 50 µ L of 0.1%w/v solution of some oxindole compounds.Timolol and melatonin were used as reference compounds.All data were obtained using 5 animals (10 eyes).One-way ANOVA and Dunn test: **-p ≤ 0.01 in comparison with melatonin; #-p ≤ 0.05 in comparison with timolol.

Figure 2 .Figure 2 .Figure 3 .
Figure 2. Graphical representation of IOP reduction after instillation of 50 µL of 0.1%w/v solution of some oxindole compounds.Timolol and melatonin were used as reference compounds.All data were obtained using 5 animals (10 eyes).One-way ANOVA and Dunn test: **-p ≤ 0.01 in comparison with melatonin; #-p ≤ 0.05 in comparison with timolol.

Figure 3 .
Figure 3. Duration of IOP decreases after the instillation (50 µL of 0.1%w/v solution per eye) of most active compounds 2. Timolol and melatonin were used as references.All data were obtained using 5 animals (10 eyes) and presented as mean ± SEM.For the «5 h» point-one-way ANOVA and Dunn test: *-p ≤ 0.05, **-p ≤ 0.01 in comparison with melatonin; #-p ≤ 0.05 in comparison with timolol.

Table 1 .
Condensation of isatins with malonic and cyanoacetic acids in different conditions.Yields of compounds 2 and 3 obtained by conventional method and under microwave irradiation.

Table 2 .
Table of optimization of microwave-assisted synthesis.
1 n/a-not active.