RU2734495C1 - Method of treating diabetes mellitus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine. Disclosed is a method of treating type 2 diabetes mellitus, providing a glucose level reduction in a body, comprising administering to a subject a therapeutically effective amount of arylidene-2-oxindole of general formula (I)

where R1 is selected from H, nitro, halogen, C1-C4-alkoxy, NH(CO)C1-C4-alkyl, NH(CO)OC1-C4-alkyl, NH(CO)C5-C6-aryl, R2 is C5- C6-aryl or C5-C6-heteroaryl containing one or more groups used in combination with each other, selected from H, OH, nitro, halogen, C1-C4-alkoxy, where C5-C6-heteroaryl means 5–6-member aryl with 1 or 2 heteroatoms selected from group O, N, S.

EFFECT: invention provides more effective reduction of blood glucose concentration by simultaneously exposing GSK3 β and α-glucosidase, that is due to simultaneous action on two glucose concentration reduction mechanisms.

7 cl, 2 tbl, 25 ex, 2 dwg

Description

Technology area

The invention relates to the fields of organic chemistry, namely the chemistry of heterocyclic compounds, medicinal chemistry and pharmacology. The proposed compounds have the ability to lower the level of glucose in the body and can be used in the treatment of type II diabetes.

State of the art

Despite significant advances in the development of hypoglycemic agents, in particular, long-acting insulin-based drugs, the problem of dysfunctions of various organs associated with an increased content of sugars in the cells of the body remains urgent. Multiple complications such as hypoglycemia, weight gain, gastrointestinal disorders, edema, etc., not only reduce the quality of life of patients, but also lead to the progression of diabetes-associated diseases, such as diabetic retinopathy and cataracts, nephropathy. Therefore, the relevance of the search for both new antidiabetic drugs and drugs that reduce the risk of diabetes-related diseases is beyond doubt. In studying the antidiabetic activity of 2-oxindole derivatives, two main strategies can be distinguished:

1) inhibition of enzymes involved in the breakdown of complex carbohydrates to easily digestible monosaccharides (α-glucosidase inhibitors);

2) reducing the risk of diabetes-related diseases caused by an increased content of sugars in the body (inhibitors of aldoreductase, glycogen synthase kinase-3β (GSK3β), as well as inhibitors of protein glycation).

It is known that 2-arylideneoxindoles are capable of inhibiting protein glycation. (Choudhary M.I., Rasheed S., Ahmed N., Khan K.M., Khan M., Rahman A., Wahab A., Anti-glycation properties of oxindole derivatives, US 2015/0038543 A1, 2015).

There are also few known ligands of kinase-3 glycogen synthase - this is an enzyme of the serine / threonine class of protein kinases involved in the insulin signaling pathway, in particular, its function is to phosphorylate glycogen synthase and to inactivate it. Inhibition of GSK3 leads to blocking access of glycogen synthase to the active site of the kinase, which in turn leads to a decrease in blood sugar levels (Frame S., Zheleva D., Expert Opinion on Therapeutic Targets, 2006, 10, 3, 429-444). Both enzyme subtypes (GSK3α and GSK3β) are expressed in insulin-sensitive peripheral tissues, and their excessive activation can also lead to insulin resistance. GSK3β hypereactivity is also associated with the onset of obesity symptoms in diabetic patients (Ring DB, Jognson KW, Henriksen EJ, Nuss JM, Goff D., Kinnick TR, Selective glycogen synthase kinase 3 inhibitors potentiate insuline activation of glucose transport and utilization in vitro and in vivo, Diabetes, 2003, 52, 588-595). A number of studies have shown that the use of low molecular weight GSK3β inhibitors leads to a significant improvement in the state of model animals with type 2 diabetes, as well as to an improvement in glycogen metabolism in the presence of a native enzyme and glycogen synthase (Coghlan M.R., Culbert A.A., Cross DA, Corcoran SL, Yates JW, Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription, Chemistry & Biology, 2000, 7, 10, 793-803).

At the moment, a limited number of synthetic ligands GSK3β based on oxindole are known, mainly indirubin derivatives. Thus, 6-bromo-substituted indirubin derivatives exhibit a significantly more selective effect with respect to GSK3β and a better inhibitory ability in comparison with unsubstituted indirubin. (Vougogiannopoulou K., Ferandin Y., Bettayeb K., Myrianthopoulos V., Lozach O., Fan Y., Johnson CH, Magiatis P., Skaltsounis A.-L., Mikros E., Meijer L., Soluble 3 ' , 6-substituted indirubins with enhanced selectivity towards glycogen synthase kinase-3 alter circadian period, Journal of Medicinal Chemistry, 2008, 51, 20, 6421-6431; Meijer L., Skaltsounis A.-L., Magiatis P., GSK- 3-Selective inhibitors derived from Tyrian Purple indirubins, Chemistry & Biology, 2003, 10, 1255-1266).

3-Arylidene-substituted derivatives of 2-oxindole are also capable of exhibiting inhibitory activity against α-glucosidase, a popular target in the treatment of type II diabetes. Studies have shown that the introduction of hydroxy groups into the 3 ', 4'-positions of the benzylidene fragment significantly enhances the affinity for α-glucosidase compared to acceptor (NO ₂ , Cl, CHO), alkoxy and alkyl groups, which indicates their role as hydrogen bond donors. An increase in the aromatic fragment itself (replacement of the benzylidene substituent with quinoline and anthracene), as well as the introduction of branched alkyl substituents in the 4'-position of the benzene ring, have a positive effect on affinity (Khan M., Yousaf M., Wadood A., Junaid M., Ashraf M ., Alam U., Ali M., Arshad M., Hussain Z., Khan K. M., Discovery of novel oxindole derivatives as potent a-glucosidase inhibitors, Bioorganic & Medicinal Chemistry, 2014, 22, 13, 3441-3448 ). This study is the closest to the claimed invention. However, the values of the half-inhibition constants of the known compounds with respect to α-glucosidase are rather high and are in the micromolar range. In addition, known compounds have not been tested in vivo, and their effect on body glucose has not been shown. No effect on GSK3β has been shown.

Disclosure of invention

To overcome these disadvantages of the known studies of the prior art, there is proposed a method for treating diabetes mellitus in a subject, providing a decrease in the level of glucose in the body, including the introduction of a composition containing a compound of general formula (I)

where R ^{1 is} selected from H, nitro, halogen, C ₁ -C ₄ -alkoxy, NH (CO) C ₁ -C ₄ -alkyl, NH (CO) OC ₁ -C ₄ -alkyl, NH (CO) C ₅ - C ₆ -aryl, NH (CO) C ₅ -C ₆ -heteroaryl, where C ₅ -C ₆ -heteroaryl means 5-6-membered aryl with 1 or 2 heteroatoms selected from the group O, N, S,

R ² means C ₅ -C ₆ -aryl or C ₅ -C ₆ -heteroaryl containing one or more groups used in combination with each other selected from H, OH, nitro, halogen, C ₁ -C ₄ -alkoxy, where C ₅ -C ₆ -heteroaryl means 5-6-membered aryl with 1 or 2 heteroatoms selected from the group O, N, S,

in a therapeutically effective amount; and a pharmaceutically acceptable carrier.

The administration of compound (I) is a single one or is carried out by a course, the frequency of administration in which and its duration are selected depending on the nature and severity of the disease or on the condition of the subject. Preferably, when the therapeutically effective amount of compound (I) is 5-50 mg / kg body weight of the subject. The administration of compound (I) is preferably carried out orally or parenterally, preferably as a composition with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is selected from the group consisting of water, buffer solutions, dimethyl sulfoxide, or mixtures thereof

In a particular embodiment of the invention, the compound of formula (I) is 3- (pyridin-2-ylmethylene) -2-oxindole or 3- (4-nitrobenzylidene) -5-methoxycarbonylamino-2-oxynol.

The invention provides the achievement of the technical result, which consists in a more effective decrease in the level of sugar in the body due to the effect on both GSK3β and α-glucosidase to varying degrees, i.e. two mechanisms are involved in reducing sugar concentration. The structure of compound (I) was optimized based on the analysis of the binding sites of the indicated targets, the selection of the optimal substituents, and the data of testing the in vitro inhibitory activity on the targets.

Brief Description of Drawings

The invention is illustrated by the following drawings.

FIG. 1 shows graphs showing the effect of oxindole derivatives (5) (A (glucose 3 g / kg), B (glucose 2 g / kg)) and (21) (C, glucose 2.5 g / kg) on blood glucose in glucose tolerance test on animal model, n = 6, * P = 0.95, ** P = 0.99, the error value is the standard error (SEM). Test compound administration - time point - 30 min, glucose administration - 0 min.

FIG. 2 shows the results of testing the cytotoxicity of oxindole derivatives in relation to PBMCs by the MTT test. * p <0.05, ** p <0.005, *** p <0.0005, **** p <0.0001

Implementation of the invention

The method of treatment within the framework of the claimed invention involves the treatment of the symptoms manifested in diabetes mellitus, namely hyperglycemia (increased glucose in the body).

The composition can be administered orally, parenterally, or by injection. In this case, the composition can be presented in the form of tablets, syrup, capsules, injections, etc. A therapeutically effective dose of the composition is selected so as to achieve a therapeutically effective concentration of the compound of formula (I) for the treatment of diabetes mellitus. In preferred embodiments of the invention, the concentration dose is selected based on two factors: the maximum possible dose is limited by the concentration of the active component that has a toxic effect, the minimum dose is limited by the concentration of the active component that causes the desired therapeutic effect - a decrease in sugar levels in the body to the required level.

Medicines can be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically).

The dose of a compound in accordance with the present invention in a subject can be adjusted depending on the therapeutic efficacy and bioavailability of the active ingredients in the body, the rate of their metabolism and excretion from the body. The dose is determined by the attending physician based on the specific parameters of the patient, such as age, body weight, sex, severity of the disease, etc. In a preferred embodiment of the invention, the active agent, a compound of formula (I), is administered at a concentration of 1-100 mg / kg, even more preferably 5-50 μ / kg of the subject's body weight.

The term "pharmaceutically acceptable" refers to a non-toxic material that does not interact with the active ingredient of the pharmaceutical composition. "Pharmaceutically acceptable carrier" refers to a biocompatible solution that is sufficiently sterile, p [Eta], isotonic, stability, and the like, and may include any solvents, diluents, including sterile saline, sodium chloride injection , Ringer's solution for injection, solution of dextrose for injection, solution of dextrose and sodium chloride for injection, lactated Ringer's solution for injection and other aqueous buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and the like. The pharmaceutically acceptable carrier may also contain stabilizers, preservatives, antioxidants, or other additives that are well known to those skilled in the art, or other excipient known in the art.

To prepare a composition with a compound of formula (I), pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, auxiliary, swelling agents, delivery devices such as preservatives, stabilizers, fillers, disintegrating agents, humectants, emulsifiers, suspending agents can be used, thickeners, sweeteners, flavors, flavors, antibacterial agents, fungicides, lubricants, delayed delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage. Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures of these substances. Protection against the action of microorganisms can be provided by a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, sorbic acid, and the like. The composition may also include isotonic agents such as sugars, sodium chloride and the like. Prolonged action of the composition can be provided by agents that delay the absorption of the active principle, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols, as well as mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate, and the like. Examples of swelling agents, wetting aids and diluents are starches, binders - alginic acid and its salts, silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. A pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous administration of an active principle, alone or in combination with another active principle, can be administered to animals and humans in a standard administration form, as a mixture with traditional pharmaceutical carriers.

Any compound falling under the structural formula (I) can be used as an active component, since the radicals R ¹ -R ³ were selected based on the data of X-ray structural analysis of complexes of indirubin with GSK3β and acarbose with α-glucosidase. These radicals provide optimal binding to selected targets, as evidenced by in vitro targeting data.

The composition implies the presence of a pharmaceutically acceptable carrier, for example, water, buffer solutions (citrate, phosphate, TRIS, etc.), dimethyl sulfoxide, ethylene glycol and other organic solvents or mixtures thereof (for a liquid composition) or cellulose derivatives, etc. (for solid composition). The solvent is selected based on the safety profile for administration to a living subject.

In some embodiments, the composition is an injection, an oral solution, a tablet, a powder (sachet), a capsule, or a syrup. The content of the compound of formula (I) can be 0-75 wt% of the total composition.

In other embodiments, the composition includes pharmaceutically compatible additives, for example, diluents, solvents, stabilizers, plasticizers, pH regulators, etc.

The possibility of implementing the claimed invention is shown, but not limited, in the examples of specific implementation.

Obtaining 3-arylidene-2-oxindoles

Example 1.3- (4-Bromobenzylidene) -5-bromo-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.3 g (0.0014 mol) of 5-bromo-2-oxindole (22b), 0.26 g (0.0014 mol) of 4-bromobenzaldehyde, 5 ml of ethyl alcohol and 75 μl piperidine (0.0008 mol). The mixture was boiled for 1 hour, then cooled to room temperature, the orange precipitate that formed was filtered off, washed with ethyl alcohol, and dried in air. The resulting product did not require additional purification. The yield was 74% (0.4 g). M.p. (exp.) = 260-262 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 6.76 ** (d, J = 8.2, 1H); 6.83 * (d, J = 8.3, 1H); 7.36 ** (d, J = 8.2, 1H); 7.40 * (d, J = 8.3, 1H); 7.48 * (s, 1H); 7.60-7.65 * (m, 3H); 7.67 ** (d, J = 8.4, 2H); 7.73 * (d, J = 8.1, 2H); 7.89 ** (s, 1H); 7.94 ** (s, 1H); 8.31 ** (d, J = 8.3, 2H); 10.78 * (s, 1H); 10.80 ** (s, 1H). * - main product, ** - minor product. According to the NMR spectrum, the ratio of the main and minor stereoisomers is 5: 3.

¹³ C NMR (DMSO-d ₆ , ppm): 111.79; 112.59; 123.26; 125.04; 131.79; 132.34; 133.16; 134.43; 136.78; 137.73; 140.35; 142.66.

IR spectrum (ZnSe, cm ^-1 ): 1446; 1604; 1701; 2854; 3014; 3049; 3082; 3118; 3163.

Mass spectrum (electron impact, 70 eV), m / z (I,%): 381 (52, М +), 380 (28, М +), 379 (100, М +), 378 (32, М + ), 351 (14), 299 (9), 272 (8), 270 (9), 224 (32), 222 (33), 191 (44), 190 (65), 164 (39), 163 (49 ), 150 (19), 149 (16), 96 (15), 75 (20), 63 (24), 50 (24), 39 (14).

Elemental analysis: found (%): C 47.51; H 2.43; N 3.69; calculated for C ₁₅ H ₉ Br ₂ NO (%): C 47.53; H 2.39; N 3.70.

Example 2. 3- (4-Bromobenzylidene) -5-methoxycarbonylamino-2-oxindole

A round-bottom flask was charged with 0.5 g (0.0024 mol) of 5-methoxycarbonylamine-2-oxindole (23i), 0.54 g (0.0029 mol) of 4-bromobenzaldehyde, 4 ml of ethyl alcohol, and 75 μl of piperidine (0, 0008 mol). The mixture was stirred at room temperature for 1 hour, then the precipitated orange precipitate was filtered off on a vacuum filter, washed with ethyl alcohol, and dried in air. The resulting product did not require additional purification. The yield was 33% (0.3 g). M.p. (exp.) = 251-253 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 3.63 (s, 3H); 6.80 (d, J = 8.4, 1H); 7.38 (d, J = 7.5, 1H); 7.53 (s, 1H); 7.65 (d, J = 8.5, 2H); 7.69 (d, J = 8.5, 2H); 7.72 (s, 1H); 9.46 (s, 1H); 10.49 (s, 1H).

¹³ C NMR (DMSO-d ₆ , ppm) 51.98; 110.53; 114.13; 121.22, 123.33; 128.74; 131.84; 132.25; 133.52; 133.92; 134.89; 138.80; 154.55; 169.04.

IR (KBr, cm ^-1 ): 1059; 1215; 1458; 1612; 1701; 1730; 3163; 3402.

Mass spectrum (electron impact, 70 eV), m / z (I,%): 374 (82, M +), 372 (82, M +), 342 (27), 340 (28), 314 (21) , 312 (18), 260 (18), 258 (14), 234 (24), 205 (43), 185 (53), 179 (42), 178 (60), 152 (36), 151 (33) , 126 (24), 102 (10), 76 (18), 59 (100), 15 (58).

Elemental analysis: found (%): C 58.18; H 3.71; N 4.22; calculated for C ₁₆ H ₁₂ BrNO ₂ (%): C 58.20; H 3.66; N 4.24.

Example 3 (E) -3- (Pyridin-2-ylmethylene) -5-bromo-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0012 mol) of 5-bromo-2-oxindole (22b), 0.14 g (0.0013 mol) of 2-pyridine-carboxaldehyde, 2 ml of methyl alcohol were introduced and 75 μl piperidine (0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of an orange powder with a yield of 67% (0.24 g). The resulting product did not require additional purification. M.p. (exp.) = 175-180 ° C (mp. (lit.) = 250-252 ° C (E-isomer), 242-246, then 250-252 (Z-isomer)).

¹ H NMR (DMSO-d ₆ , ppm, J, Hz): 6.82 (d, J = 8.3, 1H); 7.43-7.50 (m, 2H); 7.61 (s, 1H); 7.89-7.99 (m, 2H); 8.87 (d, J = 3.8, 1H); 9.27 (d, J = 1.3, 1H); 10.77 (s br, 1H).

Example 4 (E) -3- (Pyridin-2-ylmethylene) -5-nitro-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.1 g (0.0014 mol) of 5-nitro-2-oxindole (22e), 0.17 g (0.0016 mol) of 2-pyridine-carboxaldehyde, 2 ml of ethyl alcohol and 75 μl piperidine (0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of a dark gray powder with a yield of 77% (0.29 g). The resulting product did not require additional purification. M.p. (exp.) = 302 ° С (mp. (lit.)> 260 ° С (E-isomer)).

NMR ¹ H (DMSO-d ₆ , ppm, J, Hz): 7.05 (d, J = 8.6, 1H); 7.55 (pseudo t, J = 6.5, 1H); 7.74 (s, 1H); 7.97-8.03 (m, 2H); 8.24 (dd, J = 2.4, J = 8.6, 1H); 8.91 (d, J = 4.5, 1H); 10.11 (d, J = 2.2, 1H); 11.32 (s br, 1H).

Example 5. (Z) -3- (Pyridin-2-ylmethylene) -2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.1 g (0.0008 mol) of 2-oxindole (22h), 0.08 g (0.0008 mol) of 2-pyridine-carboxaldehyde, 2 ml of ethyl alcohol and 75 μl of piperidine were added (0.0008 mol). The mixture was boiled for 0.5 hour, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained as a brown powder in 79% yield (0.13 g). The resulting product did not require additional purification. M.p. (exp.) = 175-180 ° С (mp. (lit.) = 202-203 ° С [129], 196-198 ° С (Z-isomer), 205-207 ° С (Z-isomer) ).

¹ H NMR Spectrum (DMSO-d ₆ , ppm, J, Hz): 6.87 (d, J = 7.7, 1H); 6.98 (t, J = 7.6, 1H); 7.28 (t, J = 7.6, 1H); 7.46 (m, 1H); 7.57 (s, 1H); 7.86 (d, J = 7.8, 1H); 7.94 (m, 1H); 8.88 (s br, 1H); 9.00 (d, J = 7.6, 1H); 10.64 (s, 1H).

NMR ¹³ C (DMSO-d ₆ , ppm): 110.10; 121.71; 121.95; 124.60; 128.39; 128.85; 131.30; 134.14; 137.71; 144.04; 150.13; 153.67; 161.60; 169.77.

Example 6. 3- (Pyridin-3-ylmethylene) -5-bromo-2-oxindole

In a round-bottom flask equipped with a reflux condenser were introduced 0.25 g (0.0012 mol) of 5-bromo-2-oxindole (22b), 0.14 g (0.0013 mol) of 3-pyridine-carboxaldehyde, 2 ml of methanol and 75 μl piperidine (0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of a burgundy powder with a yield of 90% (0.32 g). The resulting product did not require additional purification. M.p. (exp.) = 225 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 6.79 * (d, J = 8.3, 1H); 6.85 ** (d, J = 8.3, 1H); 7.37-7.43 (m, 3H); 7.50 * (dd, J = 4.8, J = 8.0, 1H); 7.57 ** (dd, J = 4.8, J = 7.8, 1H); 7.70 ** (s, 1H), 7.96-7.97 * (m, 2H); 8.10 * (d, J = 7.9, 1H); 8.58 * (d, J = 4.4, 1H); 8.26 ** (d, J = 8.7, 2H); 8.32 * (d, J = 8.6, 2H); 8.86 ** (d, J = 1.1, 1H); 8.90 * (d, J = 8.1, 1H); 9.18 * (s, 1H); 10.76 (s br, 2H); * - main product, ** - minor product. According to the NMR spectrum, the ratio of the main and minor stereoisomers is 1.3: 1.

Mass Spectrum (HRMS-ESI): Found: 300.0006 (C ₁₄ H ₈ BrN ₂ O, M ⁺ ), calculated for C ₁₄ H ₉ BrN ₂ O: 299.9898.

Example 7. 3- (Pyridin-3-ylmethylene) -5-nitro-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0014 mol) of 5-nitro-2-oxindole (22e), 0.17 g (0.0016 mol) of 3-pyridine-carboxaldehyde, 2 ml of methanol were introduced and 75 μl piperidine (0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of a gray-green powder with a yield of 83% (0.31 g). The resulting product did not require additional purification. M.p. (exp.) = 150-155 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 7.03 * (d, J = 8.6, 1H); 7.07 ** (d, J = 8.6, 1H); 7.53 * (dd, J = 4.6, J = 7.8, 1H); 7.60 ** (dd, J = 4.9, J = 8.1, 1H); 7.85 * (s, 1H); 8.17-8.22 (m, 3H); 8.26 * (s, 1H); 8.62 ** (dd, J = 1.4, J = 4.8, 1H); 8.71-8.72 (m, 2H); 8.90-8.95 (m, 2H); 8.26 * (s, 1H); 9.24 * (d, J = 1.9, 1H); 11.41 * (s br, 1H); * - main product, ** - minor product. According to the NMR spectrum, the ratio of the major and minor stereoisomers is 1.6: 1.

Mass spectrum (HRMS-ESI): found: 266.0646 (C ₁₄ H ₈ N ₃ O ₃ , M ⁺ ) calculated for C ₁₄ H ₉ N ₃ O ₃ : 267.0644.

Example 8 3- (Pyridin-3-ylmethylene) -2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0019 mol) of 2-oxindole (22h), 0.23 g (0.0021 mol) of 3-pyridine-carboxaldehyde, 2 ml of methanol and 75 μl of piperidine ( 0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of a yellow powder with a yield of 54% (0.26 g). The resulting product did not require additional purification. M.p. (exp.) = 150-155 ° С (mp. (lit.) = 188-190 ° С).

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 6.79-6.86 ** (m, 2H); 6.98 ** (d J = 7.8, 1H); 7.19-7.24 (m, 2H); 7.33 * (s, 1H); 7.46 ** (dd, J = 5.1, J = 8.2, 1H); 7.52 * (dd, J = 4.7, J = 7.8, 1H); 7.59 ** (s, 1H); 7.69 ** (d, J = 7.8, 1H); 7.80 ** (s, 1H); 8.08 * (d, J = 7.4, 1H); 8.54 ** (dd, J = 1.6, J = 4.7, 1H); 8.61 * (dd, J = 1.2, J = 4.7, 1H); 8.84 * (s br, 1H); 9.16 ** (d, J = 2.0, 1H); 10.64 ** (s br, 1H); 10.65 ** (s br, 1H); * - main product (Z-isomer), ** - minor product (E-isomer). According to the NMR spectrum, the ratio of the major and minor stereoisomers is 1.6: 1.

Example 9.3- (Pyridin-4-ylmethylene) -5-bromo-2-oxindole (28b)

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0012 mol) of 5-bromo-2-oxindole (22b), 0.14 g (0.0013 mol) of 4-pyridine-carboxaldehyde, 2 ml of methanol were introduced and 75 μl piperidine (0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of a dark orange powder in 82% yield (0.29 g). The resulting product did not require additional purification. M.p. (exp.) = 260-265 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 6.78 ** (d, J = 8.3, 1H); 6.84 * (dd, J = 8.3, 1H); 7.32 * (d, J = 1.6, 1H); 7.40-7.44 (m, 2H); 7.61-7.63 (m, 3H); 7.90 ** (s, 1H); 7.98 ** (d, J = 1.7, 1H); 8.10 ** (d, J = 5.8, 2H); 8.67 ** (d, J = 5.9, 2H); 8.73 * (d, J = 5.8.1H); 10.85 (s br, 2H); * - main product, ** - minor product. According to the NMR spectrum, the ratio of the main and minor stereoisomers is 1.3: 1.

Mass Spectrum (HRMS-ESI): Found: 300.0004 (C ₁₄ H ₈ BrN ₂ O, M ⁺ ), calculated for C ₁₄ H ₉ BrN ₂ O: 299.9898.

Example 10 3- (Pyridin-4-ylmethylene) -5-nitro-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0014 mol) of 5-nitro-2-oxindole (22e), 0.17 g (0.0016 mol) of 4-pyridine-carboxaldehyde, 2 ml of methanol were introduced and 75 μl piperidine (0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained as a green-brown powder in 72% yield (0.27 g). The resulting product did not require additional purification. M.p. (exp.) = 260-265 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 7.01 ** (d, J = 8.7, 1H); 7.06 * (d, J = 8.7, 1H); 7.67-7.68 (m, 1H); 7.78 ** (s, 1H); 8.09 ** (d, J = 2.3, 1H); 8.11-8.13 * (m, 2H); 8.18-8.21 (m, 3H); 8.70-8.71 (m, 2H); 8.76-8.78 * (m, 2H); 11.43 (s br, 1H); * - main product, ** - minor product. According to the NMR spectrum, the ratio of the main and minor stereoisomers is 1.2: 1.

Mass spectrum (HRMS-ESI): found: 266.0648 (C ₁₄ H ₈ N ₃ O ₃ , M ⁺ ) calculated for C ₁₄ H ₉ N ₃ O ₃ : 267.0644.

Example 11 3- (Pyridin-4-ylmethylene) -2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0019 mol) of 2-oxindole (22h), 0.23 g (0.0021 mol) of 4-pyridine-carboxaldehyde, 2 ml of methanol and 75 μl of piperidine ( 0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of a light yellow powder with a yield of 76% (0.31 g). The resulting product did not require additional purification. M.p. (exp.) = 240-245 ° C (mp. (lit.) = 227-230 ° C (E-isomer), 167-171 ° C (Z-isomer)).

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 6.81-6.88 (m, 3H); 7.00 ** (t, J = 7.5, 1H); 7.23-7.27 (m, 2H); 7.35 * (d, J = 7.4, 1H); 7.54 * (s, 1H, Z-isomer); 7.62 * (d, J = 5.9, 2H); 7.72 ** (d, J = 7.4, 1H); 7.77 ** (s, 1H, E-isomer); 8.10 ** (d, J = 6.1, 2H); 8.66 ** (d, J = 6.0, 2H); 8.71 * (d, J = 5.9, 2H); 10.70 * (br s, 1H); 10.71 ** (s br, 1H); * - main product, ** - minor product. According to the NMR spectrum, the ratio of the main and minor stereoisomers is 2.7: 1.

Example 12 3- (2-Furylmethylene) -5-bromo-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0012 mol) of 5-bromo-2-oxindole (22b), 0.13 g (0.0012 mol) of furfural, 2 ml of methanol and 75 μl of piperidine ( 0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of a dark green powder with a yield of 80% (0.28 g). The resulting product did not require additional purification. M.p. (exp.) = 223-225 ° C (mp. (lit.) = 260-261 ° C).

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 6.74 * (d, J = 8.3, 1H); 6.77-6.79 ** (m, 2H); 6.82-6.84 * (m, 2H); 7.31 ** (d, J = 1.7, 1H); 7.32 ** (d, J = 3.4,1H); 7.36 ** (s, 1H); 7.39 * (s, 1H); 7.42 * (dd, J = 1.9, J = 8.3, 1H); 7.82 (s, 1H); 8.00 (d, J = 1.4, 1H); 8.25 * (s, 1H); 8.28 ** (d, J = 3.6, 1H); 8.42 * (d, J = 1.9, 1H); 10.48 ** (s br, 1H); 10.72 * (s br, 1H); * - main product, ** - minor product. According to the NMR spectrum, the ratio of the main and minor stereoisomers is 3.3: 1.

Example 13 3- (2-Furylmethylene) -5-nitro-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0014 mol) of 5-nitro-2-oxindole (22e), 0.13 g (0.0014 mol) of furfural, 2 ml of methanol and 75 μl of piperidine ( 0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained as a green-brown powder in 47% yield (0.17 g). The resulting product did not require additional purification. M.p. (exp.) = 180-185 ° C (mp. (lit.) = 305.8-306.3 ° C, 315 ° C (with decomp.)).

NMR ¹ H (DMSO-d ₆ , ppm, J, Hz): 6.87 (dd, J = 1.7, J = 3.4, 1H); 7.02 (d, J = 8.7, 1H); 7.40 (d, J = 3.6, 1H); 7.48 (s, 1H); 8.18 (dd, J = 2.3, J = 8.7, 1H); 8.28 (d, J = 1.4, 1H); 9.14 (d, J = 2.3, 1H); 11.28 (s br, 1H).

Example 14 3- (2-Furylmethylene) -2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0019 mol) of 2-oxindole (22h), 0.18 g (0.0019 mol) of furfural, 2 ml of methanol and 75 μl of piperidine (0.0008 mol ). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of a light green powder with a yield of 42% (0.17 g). The resulting product did not require additional purification. M.p. (exp.) = 180-185 ° С (mp. (lit.) = 443-446 K (E-isomer monohydrate); 170-171 ° C (Z-isomer), 178 ° C).

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 6.75 ** (dd, J = 1.8, J = 3.5, 1H); 6.79 * (dd, J = 1.8, J = 3.4, 1H); 6.82 ** (d, J = 7.9, 1H); 6.86 * (d, J = 7.7, 1H); 6.95 ** (t, J = 7.3, 1H); 7.00 * (t, J = 0.9, J = 7.6, 1H); 7.19 ** (t, J = 1.0, J = 7.6, 1H); 7.22-7.26 * (m, 2H); 7.32 * (s, 1H); 7.67 ** (s, 1H); 7.71 ** (d, J = 7.6, 1H); 7.96 ** (d, J = 1.6, 1H); 8.15 * (d, J = 1.7, 1H); 8.26 ** (d, J = 3.6, 1H); 8.36 * (d, J = 7.6, 1H); 10.57 * (s br, 1H); 10.61 ** (s br, 1H); * - main product (E-isomer), ** - minor product (Z-isomer). According to the NMR spectrum, the ratio of the main and minor stereoisomers is 9: 1.

Example 15 3- (2-Thienylmethylene) -5-bromo-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0012 mol) of 5-bromo-2-oxindole (22b), 0.13 g (0.0012 mol) of thiophene-2-carboxaldehyde, 2 ml of methanol were introduced and 75 μl piperidine (0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained in the form of a bright yellow powder with a yield of 43% (0.16 g). The resulting product did not require additional purification. M.p. (exp.) = 275-280 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz) 6.80 * (d, J = 8.3, 1H); 6.83 ** (d, J = 8.3, 1H); 7.25 * (dd, J = 3.7, J = 5.1, 1H); 7.34 * (dd, J = 2.0, J = 8.3, 1H); 7.46 ** (t, J = 1.9, J = 8.4, 1H); 7.86-7.87 ** (m, 3H); 7.91-7.94 * (m, 3H); 9.05 ** (d, J = 5.2, 1H); 8.26 * (s, 1H); 10.73 * (s br, 1H); 10.78 ** (s br, 1H); * - main product, ** - minor product. According to the NMR spectrum, the ratio of the main and minor stereoisomers is 5: 1.

Mass Spectrum (HRMS-ESI) Found: 304.9516 (C ₁₃ H ₇ BrNOS, M ⁺ ), calculated for C ₁₃ H ₈ BrNOS: 304.9510.

Example 16. 3- (2-Thienylmethylene) -2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0019 mol) of 2-oxindole (22h), 0.22 g (0.0021 mol) of thiophene-2-carboxaldehyde, 2 ml of methanol and 75 μl of piperidine ( 0.0008 mol). The mixture was boiled for 5 hours, then cooled to room temperature, the excess solvent was removed in a vacuum rotary evaporator. The product was obtained as a dark yellow powder with a yield of 67% (0.28 g). The resulting product did not require additional purification. M.p. (exp.) = 212-216 ° C (mp. (lit.) = 463-466 K (Z-isomer); 160-162 ° C).

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 6.84 * (d, J = 7.6, 1H); 6.90 ** (d, J = 7.6, 1H); 6.98 * (t, J = 7.6, 1H); 7.04 ** (d, J = 7.5, 1H); 7.19 * (t, J = 7.7, 1H); 7.22 * (dd, J = 3.7, J = 5.0, 1H); 7.25-7.31 ** (m, 2H); 7.67 * (d, J = 7.5, 1H); 7.77 ** (s, 1H), 7.81 ** (d, J = 3.5, 1H); 7.88 * (d, J = 5.1, 1H); 7.92 * (d, J = 3.4,1H); 7.98 ** (d, J = 5.3, 1H); 8.10 * (s, 1H); 8.16 ** (d, J = 7.8, 1H); 10.60 * (s br, 1H); 10.62 ** (s br, 1H); * - main product (Z-isomer), ** - minor product (E-isomer). According to the NMR spectrum, the ratio of the main and minor stereoisomers is 9: 1.

Example 17 3- (4-Hydroxybenzylidene) -5-acetamido-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.1 g (0.0006 mol) of 5-acetamido-2-oxindole (24e), 0.07 g (0.0006 mol) of 4-hydroxybenzaldehyde, 3 ml of ethyl alcohol and 75 μl piperidine (0.0008 mol). The mixture was boiled for 0.5 hour, then cooled to room temperature. The solvent was removed in vacuo. The resulting product did not require additional purification. The yield was 100% (0.17 g). M.p. (exp.) = 190-195 ° C.

¹ H NMR (DMSO-d ₆ , ppm, J, Hz): 1.99 (s, 3H); 6.78 (d, J = 8.3, 1H); 6.87 (d, J = 8.6, 2H); 7.39 (dd, J = 1.9, J = 8.3, 1H); 7.51 (s, 1H); 7.62 (d, J = 8.7, 2H); 8.13 (d, J = 1.9, 1H); 9.81 (m, 2H).

¹³ C NMR (DMSO-d ₆ , ppm): 24.29; 109.98; 114.47; 116.46; 120.83; 121.84; 124.45; 124.58; 132.57; 133.56; 137.40; 138.44; 161.08; 168.27; 167.79.

IR spectrum (KBr, cm ^-1 ): 1477; 1566; 1599; 1666; 1680; 2590-3622; 2854; 2927; 3269; 3419.

Elemental analysis: found (%): C 69.35; H 4.85; N 9.50; calculated for C ₁₇ H ₁₄ N ₂ O ₃ (%): C 69.38; H 4.79; N 9.52.

Example 18 3- (4-Hydroxybenzylidene) -5-methoxycarbonylamino-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.3 g (0.0015 mol) of 5-methoxycarbonylamine-2-oxindole (23i), 0.18 g (0.0015 mol) of 4-hydroxybenzaldehyde, 5 ml of ethanol and 75 μl piperidine (0.0008 mol). The mixture was boiled for 0.5 hour, then cooled to room temperature, the precipitate that formed was filtered off on a vacuum filter, washed with ethyl alcohol. The filtrate was concentrated in a rotary evaporator vacuum. It was washed with concentrated hydrochloric acid, then with water, and dried in air. The resulting product did not require additional purification. The yield was 44% (0.2 g). M.p. (exp.) = 205-210 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 3.62 (s, 3H); 6.78 (d, J = 8.3, 1H); 6.90 (s, 1H); 6.92 (s, 1H); 7.28 (d, J = 8.0, 1H); 7.51 (s, 1H); 7.62 (s, 1H); 7.64 (s, 1H); 7.98 (s, 1H); 9.50 (s, 1H); 10.43 (s, 1H).

¹³ C NMR (DMSO-d ₆ , ppm) 51.98; 110.19; 116.20; 120.40; 121.89; 125.04; 125.31; 132.45; 135.34; 137.19; 138.27; 154.63; 159.81; 160.66.

IR (KBr, cm ^-1 ): 1165; 1215; 1367; 1446; 1639; 1697; 2500-3500.

Mass spectrum (electron impact, 70 eV), m / z (I,%): 310 (100, М +), 278 (43), 250 (33), 221 (11), 196 (22), 185 ( 12), 151 (7), 115 (8), 77 (4), 59 (22), 15 (9).

Elemental analysis: found (%): C 71.88; H 4.95; N 5.23; calculated for C ₁₆ H ₁₃ NO ₃ (%): C 71.90; H 4.90; N 5.24.

Example 19 3- (4-Hydroxybenzylidene) -5-benzoylamino-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.1 g (0.0004 mol) of 5-benzoylamino-2-oxindole (23j), 0.05 g (0.0004 mol) of 4-hydroxybenzaldehyde, 3 ml of ethyl alcohol and 75 μl piperidine (0.0008 mol). The mixture was boiled for 1 hour, then cooled to room temperature, the solvent was removed in vacuo. The dry residue was washed with chloroform and dried in air. The resulting product did not require additional purification. The yield was 99% (0.15 g). M.p. (exp.) = 179-180 ° C.

¹ H NMR (DMSO-d ₆ , ppm, J, Hz): 5.60 (s br, 1H); 6.91 (d, J = 8.6, 2H); 7.48-7.60 (m, 5H); 7.67 (d, J = 8.4, 2H); 7.91 (d, J = 7.1, 1H); 8.31 (s, 1H); 10.18 (s, 1H); 10.54 (s br, 1H).

¹³ C NMR (DMSO-d ₆ , ppm): 110.02; 115.75; 115.97; 116.26; 122.61; 124.91; 125.26; 128.05; 128.79; 128.84; 131.82; 132.51; 133.26; 135.41; 137.28; 160.01; 165.76; 169.79.

IR spectrum (KBr, cm ^-1 ): 1477; 1579; 1599; 1643; 1685; 1763-3685; 2812; 2949; 3157.

Mass spectrum (electron impact, 70 eV), m / z (I,%): 356 (47, М +), 251 (8), 196 (12), 167 (4), 151 (3), 105 ( 100), 84 (7), 77 (53), 51 (8), 27 (6).

Elemental analysis: found (%): C 74.14; H 4.55; N 7.85; calculated for C ₂₂ H ₁₆ N ₂ O ₃ (%): C 74.15; H 4.53; N 7.86.

Example 20 3- (4-Hydroxybenzylidene) -5-furoylamino-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.1 g (0.0004 mol) of 5-furoylamino-2-oxindole (23k), 0.04 g (0.0004 mol) of 4-hydroxybenzaldehyde, 3 ml of ethyl alcohol and 75 μl piperidine (0.0008 mol). The mixture was boiled for 1 hour, then cooled to room temperature, the solvent was removed in vacuo. The dry residue was washed with chloroform and dried in air. The resulting product did not require additional purification. The yield was 87% (0.12 g). M.p. (exp.) = 187-190 ° C.

NMR ¹ H (DMSO-d ₆ , ppm, J, Hz): 6.67 (s, 1H); 6.91 (d, J = 8.1, 1H); 7.27-7.34 (dd, J = 3.1, J = 12.4, 2H); 7.50-7.56 (m, 2H); 7.66 (d, J = 8.3, 2H); 7.88-7.98 (m, 2H); 8.31 (s, 1H); 10.12 (s, 1H); 10.55 (s br, 1H).

¹³ C NMR (DMSO-d ₆ , ppm): 110.04; 112.53; 113.28; 114.83; 115.78; 116.00; 116.27; 122.66; 125.23; 132.55; 135.42; 137.36; 139.22; 145.92; 156.48; 160.04; 168.04; 169.76.

IR spectrum (KBr, cm ^-1 ): 175; 1587; 1655; 1685; 2526-3627; 2949; 3215; 3394.

Mass spectrum (electron impact, 70 eV), m / z (I,%): 346 (100, М +), 318 (3), 289 (3), 251 (20), 242 (10), 223 ( 9), 196 (19), 167 (6), 147 (3), 95 (48), 56 (2), 39 (9).

Elemental analysis: found (%): C 69.35; H 4.12; N 8.07; calculated for C ₂₀ H ₁₄ N ₂ O ₄ (%): C 69.36; H 4.07; N 8.09.

Example 21 3- (4-Nitrobenzylidene) -5-methoxycarbonylamino-2-oxynol

In a round-bottom flask equipped with a reflux condenser, 0.2 g (0.001 mol) of 5-methoxycarbonylamine-2-oxindole (23i), 0.15 g (0.001 mol) of 4-nitroxybenzaldehyde, 3 ml of ethyl alcohol and 75 μL of piperidine (0 , 0008 mol). The mixture was boiled for 0.5 hour, then cooled to room temperature, the precipitate formed was filtered off, washed with ethyl alcohol, and dried in air. The resulting product did not require additional purification. The yield was 49% (0.16 g). M.p. (exp.) = 265-267 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 3.59 * (s, 3H); 3.66 ** (s, 3H); 6.76 ** (d, J = 8.4, 1H); 6.80 * (d, J = 8.4, 1H); 7.20 ** (d, J = 8.1, 1H); 7.42 * (d, J = 7.1, 1H); 7.56 * (s, 1H); 7.63 * (s, 1H); 7.76 ** (s, 1H); 7.84 ** (s, 1H); 7.93 * (d, J = 8.6, 2H); 8.26 ** (d, J = 8.7, 2H); 8.32 * (d, J = 8.6, 2H); 8.50 ** (d, J = 8.7, 2H); 9.47 * (s br, 1H); 9.52 ** (br s, 1H); 10.59 * ^, ** (br.s, 2H). * - main product, ** - minor product. According to the NMR spectrum, the ratio of the main and minor stereoisomers is 3: 1.

¹³ C NMR (DMSO-d ₆ , ppm) 51.98; 110.73; 120.85; 121.62; 123.49; 124.37; 130.80; 130.92; 133.02; 133.41; 139.15; 147.79; 154.48; 168.66.

IR spectrum (KBr, cm ^-1 ): 1315; 1352; 1460; 1506; 1709; 1730; 2841; 3035; 3076; 3116; 3168; 3384.

Mass spectrum (electron impact, 70 eV), m / z (I,%): 339 (100, М +), 307 (80), 280 (20), 261 (16), 234 (17), 205 ( 17), 178 (15), 151 (8), 126 (5), 76 (10), 59 (58), 15 (60).

Elemental analysis: found (%): C 60.16; H 3.91; N 12.37; calculated for C ₁₇ H ₁₃ N ₃ O ₅ (%): C 60.18; H 3.86; N 12.38.

Example 22 3- (3,4,5-Trimethoxybenzylidene) -5-acetamido-2-oxoindole

A round-bottom flask was charged with 0.47 g (0.003 mol) of 5-acetamide-2-oxindole (24e), 0.59 g (0.003 mol) of 3,4,5-trimethoxybenzaldehyde, 2 ml of ethyl alcohol and 75 μL of piperidine (0, 0008 mol). The mixture was stirred at room temperature for 4 hours, then the precipitated yellow precipitate was filtered off on a vacuum filter, washed with water, and dried in air. The resulting product did not require additional purification. The yield was 43% (0.40 g). M.p. (exp.) = 245-246 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 1.98 (s, 3H); 3.37 (s, 1H); 3.75 (s, 3H); 3.83 (s, 6H); 6.81 (d, J = 3.4, 1H); 7.05 (s, 2H); 7.35 (d, J = 3.8, 1H); 7.56 (s, 1H); 8.26 (s, 1H); 9.80 (s, 1H); 10.47 (s, 1H).

¹³ C NMR (DMSO-d ₆ , ppm): 24.25; 56.25; 60.61; 107.45; 110.33; 114.85; 121.44; 121.50; 127.17; 129.97; 133.82; 136.81; 139.03; 153.37; 168.32; 169.45.

IR (liquid paraffin, cm ^-1 ): 1150; 1720; 3185; 3260.

Elemental analysis: found (%): C 65.29; H 5.61; N 7.64; calculated for C ₂₀ H ₂₀ N ₂ O ₅ (%): C 65.21; H 5.47; N 7.60.

Example 23 3- (3,4,5-Trimethoxybenzylidene) -5-furoylamino-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.1 g (0.0003 mol) of 5-furoylamino-2-oxindole (23k), 0.08 g (0.0004 mol) of 3,4,5-trimethoxybenzaldehyde, 3 ml ethyl alcohol and 75 μl piperidine (0.0008 mol). The mixture was boiled for 2 hours, then cooled to room temperature. To the reaction mixture was added 10 ml of cold water, the orange precipitate was filtered off, washed with cold water, and dried in air. The resulting product did not require additional purification. The yield was 52% (0.09 g). M.p. (exp.) = 258-262 ° C.

¹ H NMR spectrum (DMSO-d ₆ , ppm, J, Hz): 3.75 (s, 3H); 3.82 (s, 6H); 6.66 (s, 1H); 6.86 (d, J = 8.2, 1H); 7.08 (s, 1H); 7.25 (d, J = 2.2, 1H); 7.50-7.60 (m, 2H); 7.89 (s, 1H); 8.40 (s, 1H); 10.11 (s, 1H); 10.55 (s, 1H).

¹³ C NMR (DMSO-d ₆ , ppm): 56.26; 60.63; 107.58; 110.34; 110.73; 112.53; 114.83; 116.22; 123.05; 127.04; 129.93; 132.83; 136.98; 137.91; 139.66; 145.99; 148.01; 153.39; 156.48; 169.48.

IR spectrum (ZnSe, cm ^-1 ): 1456; 1475; 1491; 1574; 1587; 1608; 1645; 1676; 2833; 2935; 3251.

Mass spectrum (electron impact, 70 eV), m / z (I,%): 420 (92, М +), 405 (9), 373 (6), 319 (3), 295 (10), 249 ( 6), 224 (10), 196 (5), 141 (6), 95 (100), 67 (4), 39 (8).

Elemental analysis: found (%): C 65.69; H 4.83; N 6.65; calculated for C ₂₃ H ₂₀ N ₂ O ₆ (%): C 65.71; H 4.79; N 6.66.

Example 24 3- (3,4,5-Trimethoxybenzylidene) -5- (N-methyl-N'-thiocarbonylamino) -2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.15 g (0.0007 mol) of N-Methyl-N '- (2-oxidol-3-yl) thiourea (23p), 0.13 g (0.0007 mol) 3,4,5-trimethoxybenzaldehyde, 2 ml of ethyl alcohol and 75 μl of piperidine (0.0008 mol). The mixture was boiled for 1 hour, then cooled to room temperature, the precipitate that formed was filtered off with a paper filter. The filtrate was concentrated in a rotary evaporator vacuum. The product was purified by column chromatography (eluent EtOAc). The yield was 26% (0.07 g). M.p. (exp.) = 220-225 ° C.

¹ H NMR spectrum (CDCl ₃ , δ, ppm, J Hz): 2.83 * (d, J = 4.4, 3H); 2.89 ** (d, J = 4.3, 3H); 3.74 * (s, 3H); 3.74 ** (s, 3H); 3.80 * (s, 6H); 3.84 ** (s, 6H); 6.82 ** (d, J = 8.1, 1H); 6.86 * (d, J = 8.2, 1H); 7.01 ** (d, J = 8.1, 1H); 7.05 * (s, 2H); 7.10 * (dd, J = 1.6, J = 8.3, 1H); 7.38 ** (s br, 1H); 7.41 * (s br, 1H); 7.56 ** (s, 1H); 7.59 * (s, 1H); 7.67 * (d, J = 1.6, 1H); 7.79 ** (s, 1H); 8.06 ** (s, 2H); 9.31 * (s br, 1H); 9.39 ** (s br, 1H); 10.63 * ^, ** (s, 2H). * - main product, ** - minor product. According to the NMR spectrum, the ratio of the main and minor stereoisomers is 3: 2.

¹³ C NMR (DMSO-d ₆ , ppm): 56.32; 56.47; 60.67; 107.58; 110.74; 116.85; 117.58; 127.03; 128.05; 130.00; 137.13; 140.33; 141.08; 147.21; 152.72; 153.38; 169.39; 182.12.

IR (KBr, cm ^-1 ): 1126; 1244; 1309; 1427; 1464; 1479; 1697; 1712; 2343; 2360; 2835; 2935; 3070; 3192.

Mass spectrum (electron impact, 70 eV), m / z (I,%): 368 (100, М +), 353 (20), 326 (22), 267 (12), 209 (7), 181 ( 5), 153 (4), 119 (3), 73 (2), 30 (4).

Mass spectrum (HRMS-ESI): found: 400.1326 (C ₂₀ H ₂₁ N ₃ O ₄ S, M + H), 422.1145 (C ₂₀ H ₂₁ N ₃ O ₄ S, M + Na), 438 , 0884 (C ₂₀ H ₂₁ N ₃ O ₄ S, M + K), calculated for C ₂₀ H ₂₁ N ₃ O ₄ S: 399.1253.

Example 25 3- (3-Hydroxybenzylidene) -5-acetamido-2-oxindole

In a round-bottom flask equipped with a reflux condenser, 0.25 g (0.0013 mol) of 5-acetamido-2-oxindole (24e), 0.16 g (0.0013 mol) of 3-hydroxybenzaldehyde, 4 ml of ethyl alcohol and 75 μl piperidine (0.0008 mol). The mixture was boiled for 0.5 hour, then cooled to room temperature, the precipitate was filtered off on a paper filter, the filtrate was concentrated in a vacuum rotary evaporator. The product was purified using flash chromatography: the first fraction — EtOAc eluent, contains unreacted aldehyde; the second fraction - eluent EtOAc: MeOH (1: 1), contains the product. The solvent was removed in vacuo. The yield was 52% (0.2 g). M.p. (exp.) = 285-287 ° C.

¹ H NMR Spectrum (DMSO-d ₆ , ppm, J, Hz): 1.97 (s, 3H); 6.80 (d, J = 8.3, 1H); 6.87 (d, J = 8.1, 1H); 7.06 (s, 1H); 7.14 (d, J = 7.6, 1H); 7.31 (t, J = 7.7, 1H); 7.47 (d, J = 8.5, 1H); 7.51 (s, 1H); 7.95 (s, 1H); 9.75 (s br, 1H); 9.84 (s, 1H); 10.52 (s, 1H).

¹³ C NMR (DMSO-d ₆ , ppm): 24.22; 110.22; 115.33; 116.35; 117.46; 120.51; 121.28; 122.02; 127.93; 130.36; 133.64; 135.84; 136.59; 139.04; 158.07; 168.26; 169.33.

IR (KBr, cm ^-1 ): 1257; 1377; 1446; 1568; 1664; 1695; 3165; 3228; 3298; 2900-3400.

Mass spectrum (electron impact, 70 eV), m / z (I,%): 294 (100, М +), 252 (81), 224 (32), 196 (13), 159 (9), 115 ( 4), 77 (2), 43 (28).

Elemental analysis: found (%): C 69.36; H 4.85; N 9.50; calculated for C ₁₇ H ₁₄ N ₂ O ₃ (%): C 69.38; H 4.79; N 9.52.

In vitro measurement of GSK3β and α-glucosidase inhibitory activity

The activity of GSK3β (human active recombinant) and α-glucosidase (murine) was determined by the bioluminescence method using the ADP-Glo ™ KinaseAssay kit (Promega, USA). The assay was performed at 27 ° C in a 96-well flat-bottomed white plate (Greiner Lumitrac 655074, USA) in a final incubation volume of 25 μl.

For kinase-3β glycogen synthase, the incubation mixture contained 2 ng / ml kinase, 0.2 mg / ml substrate GSK3β YRRAAVPPSPSLSRHSSPHQ (pS) EDEEE obtained from human muscle glycogen synthase type 1 (amino acids 636-661) and 25 μM ATP solution in buffer containing 40 mM Tris-HCl (pH 7.5), 20 mM MgCl ₂ , 0.1 mg / ml BSA, 50 μm DTT ((-) control). Then a 1.25% solution of the tested compounds was added in DMSO at concentrations of 1⋅10 ^-4 -1⋅10 ^-8 (at least 3 parallel measurements) and incubated in a thermostated shaker PST-60HL (Biosan, Latvia) for 60 min. [231, 232]. Luminescence was measured using an Infinite M200 PRO microplate reader (Tecan, Austria), integration time 1000 ms.

For α-glucosidase (EC 3.2.1.20 expressed by S. cerevisiae, Sigma # G5003), a protein solution at a concentration of 0.12 U / ml was incubated with the test compound in 0.1 M phosphate buffer (pH 6.8) at 37 ° C. within 5 minutes. Then, 25 μl of a 4 mM substrate solution - p-nitrophenyl-α-D-glucopyranoside (Sigma # N1377) was added to the incubation mixture, and the absorption spectrum was recorded at 400 nm for 15 minutes using an Infinite M200 PRO microplate reader (Tecan, Austria) [ 233].

An experimental ATP-competitive inhibitor GSK3β SB-216763 (Sigma, USA) [234] and a standard α-glucosidase inhibitor, acarbose [13], were used as (+) controls. The concentration value causing 50% inhibition of enzyme activity was determined based on the concentration dependences of the tested enzyme activity using the OriginPro 9.0 software (Table 2).

For a number of benzylidene-substituted ones, we further evaluated the in vitro inhibitory ability towards kinase-3β glycogen synthase. The experimental ATP-competitive inhibitor GSK3β - 3- (2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione (SB- 216763, Table 1).

In vivo study of antidiabetic activity

The measurement was carried out on male Wistar rats aged 9-12 weeks. The animals were kept at 4 rats per cage, with free access to water and food, a 12/12 h / h day / night cycle at a temperature of 25 ° C. All animal studies were approved by the Animal Care and Use Committee of the Volgograd State Medical University (Volgograd State Medical University).

Diabetes induction

Prior to testing, animals were fed a balanced, high-fat diet (58% fat, 25% protein, 17% carbohydrates,% total calories) for 2 weeks. Then the animals were divided into 2 groups: the experimental group of animals received intraperitoneal administration of a solution of streptozotocin in citrate buffer pH 4.4 (35 mg / kg), the control group - citrate buffer (1 ml / kg). One week after administration, the animals were weighed and blood glucose was determined to confirm the induction of diabetes. Animals with glucose levels above 15 mM were used for further testing.

Glucose tolerance test

The animals were divided into three groups (n = 6 for animals with induced diabetes; n = 5 without induction of diabetes): experimental animals treated with the test drug or reference substance, and the control group.

Vildagliptin in 0.5% sodium carboxymethylcellulose solution was used as a (+) control; as (-) control - the equivalent volume of the carrier (0.5% sodium carboxymethylcellulose solution).

To carry out the glucose-tolerance test, the animals were not fed overnight, then at a time point of 30 minutes, the test compound was administered intraperitoneally (5 or 50 mg / kg) in 0.5% sodium carboxymethylcellulose solution (group 1), 10 mg / kg reference drug ((+) - control, group 2) and carrier solution (group 3). After 30 minutes, all groups received intragastric administration of a glucose solution (2, 2.5 or 3 g / kg) in isotonic sodium chloride solution (0.9%). To control the glucose level, venous blood was taken from the tail of each animal (20 μl) every 30 minutes from the beginning of the administration of the test substances for 2.5 hours. Blood samples were hemolyzed with 1 ml of glucose / lactate hemolytic solution. The glucose level was determined using a Biosen C_Line biochemical analyzer (EKF Diagnostics, Germany).

An in vivo test of compounds (5) and (21) in a glucose tolerance test was carried out in animal model mice with induced diabetes (Fig. 1). A highly selective dipeptidyl peptidase-4 inhibitor vildagliptin (II), which is used in the treatment of type 2 diabetes mellitus, was used as a comparison compound.

A group of animals treated with vehicle was used as a negative control.

According to the results of an in vivo experiment, it can be seen that oxindole derivatives cause a decrease in blood glucose levels after glucose administration (2 and 2.5 g / kg) to normal levels, which indicates the effectiveness of these compounds in the treatment of diabetes. An improved effect of compound 21) was found in comparison with vildagliptin - an effective reduction in blood sugar was achieved using a dose of the test substance half that of the comparison compound.

Despite the fact that in the specific examples two specific concentrations of compound (I) are considered, it is clear to the person skilled in the art that when other substituents R ¹ -R ^{3 are used,} these concentrations will be different depending on the activity of the particular compound. It is also clear to the person skilled in the art that a specific dose can be adjusted for each specific compound based on target activity data and toxicity data.

Measurement of cytotoxicity in vitro

Human A549 cell cultures were grown in DMEM medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% gentamicin as an antibiotic at 37 ° C and 5% CO ₂ in a humid atmosphere.

The cytotoxicity of the synthesized compounds was determined by the MTT test. The cells were seeded at a concentration of 1 × 10 ⁴ cells / 200 μl in a 96-well plate and cultured at 37 ° C in a humidified atmosphere with 5% CO ₂ . After 24 hours of incubation, various concentrations (at least 3 parallel measurements) of the tested compounds (from 100 to 1.56 μM / L) were added to the cell cultures, and then the cells were cultured under the same conditions for 72 hours. All substances were dissolved in DMSO, the final concentration of DMSO in the well did not exceed 0.1% and was not toxic to cells. As a (-) control, a solvent with a final concentration of DMSO of 0.1% was used. After incubation, 20 μl MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide, 5 mg / ml) was added to each well and the plates were incubated for an additional 2 hours. Next, the medium was removed from the plates and 100 μl of DMSO was added to each well to dissolve the formed formazan crystals. Using a plate analyzer (Victor3, PerkinElmer), the absorbance was determined at 530 nm, minus the measured background absorbance at 620 nm. The concentration value causing 50% inhibition of cell population growth (IC50) was determined from dose-response curves using OriginPro 9.0 software.

Isolation of PBMCs

To isolate PBMCs, venous blood (20 ml each) was collected from various healthy non-smoking young donors into sterile heparin-containing tubes. Blood was diluted with the same volume of phosphate buffer, mixed gently by inversion and layered on 5 ml Ficoll (Ficoll-PaqueTM Plus, GE Healthcare Bio-Sciences AB) in 15 ml plastic tubes (4 tubes). The contents of the tubes were centrifuged at 2000 rpm and room temperature for 30 minutes. The supervascular fluid was removed, and the mononuclear cell layer was collected, transferred into other 15 ml tubes and washed three times with phosphate buffer by centrifugation at 1000 rpm and room temperature for 10 minutes. The supernatant was removed, the precipitated cells were resuspended in 1 ml in DMEM (Dubelko's medium or double modification of Eagle's medium, Gibco by life technologiesTM, UK). A hemocytometer was used to count the cells and assess their viability. Cell viability, determined by 0.4% trypan blue (Sigma-Aldrich, Co., St. Louis, USA), was> 95% in the experiment. Cell concentration was adjusted to 1.0 x 10 ⁶ cells / ml with Turk staining solution based on morphological criteria in final DMEM medium (Gibco) supplemented with 2 mM L-glutamine (Gibco) 10% heat inactivated fetal bovine serum (BioClot, Germany), penicillin (100 IU / ml) and streptomycin (100 mg / ml) (Gibco), and then plated into 96-well plates (SPL Life Sciences Co., Ltd., Korea) 0.2 ml each / hole. The cell culture was left for 1 h at 37 ° C in a humidified atmosphere containing 5% CO ₂ . Then the wells were washed to remove non-adhesive cells, then filled with 0.2 ml of the final medium. After 24 hours of incubation, the cells were used to measure the cytotoxicity of the test compounds.

Measurement of cytotoxicity on PBMCs

Cytotoxicity was measured by the MTT test method based on mitochondria-dependent reduction of 3- (4,5-dimethylthiazol-2-yl) -5-diphenyltetrazolium bromide (MTT) to fomazan. For this, PBMCs cells (2.0 × 10 ⁵ per well) were used, grown in 96-well plates, isolated as described above. For test compounds and reference samples, 4 m well data were used. Test compounds were dissolved in DMSO (to a concentration of 0.1 M), filtered through disposable filters (pore diameter 0.20 μm, Corning, NY, Germany), and adjusted to concentrations of 1 × 10 ^-5 , 1 × 10 ^-6 , 1 × 10 ^-7 M using phosphate buffer (final concentration of DMSO 0.5%). As (-) - control we used DMEM medium and DMEM medium containing 0.5% DMSO. As an internal control for the activation of mononuclear cells, we used a medium containing 10 μg / ml of lipopolysaccharides (LPS) from E. coli O111: B4 (Sigma-Aldrich, St. Louis, USA), and as a (+) control, a medium containing 10 % Triton X-100.

After addition of the test compound, the cells were incubated for 24 hours at 37 ° C in a humidified atmosphere containing 5% CO ₂ . MTT (Sigma, St. Louis, MO, USA) was dissolved in phosphate buffer (5 mg / ml), followed by sterilization filtration. At the end of the incubation time, 20 μL of MTT solution was added to each well, then the resulting solutions were incubated at 37 ° C in a humidified atmosphere containing 5% CO _{2 for} another 4 hours. After 4 hours, the incubation medium was removed, 150 were added to each well. μl of DMSO to dissolve fomazan crystals. The microplates were shaken at room temperature for 10 minutes, after which the optical density of the solutions was recorded at a wavelength of 540 nm using a Sunrise TS microplate reader (Tecan GmbH, Austria). Cytotoxicity was calculated as the percentage of the mean optical density at each concentration of the test compound (n = 4) in relation to untouched cells.

Was tested cytotoxicity of a number of synthesized derivatives in relation to blood cells PBMCs (Fig. 2).

All tested compounds show satisfactory toxicity on PBMC cells. In particular, we have shown that the leader compound with respect to GSK3β, identified in the course of this work, has moderate cytotoxicity in a cellular assay with a sufficient therapeutic window.

Thus, the application of arylidene-2-oxindole of general formula (I) for the treatment of diabetes mellitus has been demonstrated.

Claims (10)

1. A method of treating diabetes mellitus of the second type, providing a decrease in glucose levels in the body, comprising administering to a subject a therapeutically effective amount of arylidene-2-oxindole of general formula (I)

where R1 is selected from H, nitro, halogen, C1-C4-alkoxy, NH (CO) C1-C4-alkyl, NH (CO) OC1-C4-alkyl, NH (CO) C5-C6-aryl, R2 means C5-C6-aryl or C5-C6-heteroaryl containing one or more groups used in combination with each other selected from H, OH, nitro, halogen, C1-C4-alkoxy, where C5-C6-heteroaryl means 5 -6-membered aryl with 1 or 2 heteroatoms selected from the group O, N, S. 2. The method according to claim 1, characterized in that the administration is one-time or is carried out by a course, the frequency of administration and its duration are selected depending on the nature and severity of the disease or on the condition of the subject. 3. The method according to claim 1, characterized in that the therapeutically effective amount of compound (I) is 5-50 mg / kg body weight of the subject. 4. A process according to claim 1, characterized in that the compound of formula (I) is 3- (pyridin-2-ylmethylene) -2-oxindole. 5. A process according to claim 1, characterized in that the compound of formula (I) is 3- (4-nitrobenzylidene) -5-methoxycarbonylamino-2-oxynol. 6. The method according to claim 1, characterized in that the administration is carried out orally or parenterally. 7. A method according to claim 1, characterized in that the compound of formula (I) is administered in combination with a pharmaceutically acceptable carrier or excipient.

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