RU2039733C1 - Derivatives of 2-(3,4-dihydroxyphenyl) ethylamine showing immunotropic activity and capability to inhibit virus replication - Google Patents

Abstract

FIELD: organic chemistry. SUBSTANCE: product of the formula [2-R¹O-3-R²O]Ph-(CH₂)₂-NHR² where R¹R² a group -C(O)-Ph-NO₂ -C(O)-CH(CH₃)₂ -C(O)-CH(C₃H₇)₂ -C(O)-C₆H₁₁ -C(O)-CH₂-C₆H₁₁ -C(O)-CH₂-C₅H₉ -C(O)-CH₂-Cl -C(O)-(CH₂)₃-Cl -C(O)-(CH₂)₄-Cl SO₂C₆H₄CH₃ -S(O)-O-Ph-Cl; -C(O)-Ad, where Ad 1-adamantyl, or R¹ hydrogen and R² as indicated above. Reagent 1: dopamine hydrochloride. Reagent 2: chloroanhydride of corresponding acid. Reaction condition: in pyridine medium, at 5-10 C. Derivatives were in substituted amine chemistry. EFFECT: improved method of synthesis. 3 cl, 6 tbl

Description

 The invention relates to new chemical compounds, specifically to derivatives of 2- (3,4-dihydroxyphenyl) ethylamine, which exhibit immunotropic activity and have the ability to inhibit the replication of the virus.

 Known structural analogues of the claimed compounds with other types of activity. Thus, the drug ibopamine, which has antispasmodic properties, is used as a cardiovascular agent [1] and ethyl ester of 3,4-dicarboxy-β-phenethylcarbamic acid exhibits antidepressant [2] and antiparkinsonian [3] activity. Immunotropic activity and the ability to inhibit virus replication among structural analogues of the claimed compounds are unknown.

Currently, the drug levamisole [4] whose drawback is its rather high toxicity (LD ₅₀ is 50 mg / kg), is widely used to treat diseases associated with immune disorders.

 The aim of the invention are derivatives of 2- (3,4-dihydroxyphenyl) ethylamine, exhibiting immunotropic activity, the ability to inhibit the replication of the virus and having low toxicity.

CH₂CH₂NHR² где R¹=R² C(O)C₆H₄NO₂, -C(O)CH(CH₃)₂, -C(O)C₆H₁₁, -C(O)CH₂C₆H₁₁, -C(O)CH₂C₅H₉, -C(O)(CH₂)₄Cl, -C(O)(CH₂)₃Cl, -C(O)CH₂Cl, -C(O)-CH(C₃H₇)₂, -SO₂C₆H₄CH₃, -SO₂C₆H₄Cl,

или R¹=H, a R² принимает указанные значения.This goal is achieved by derivatives of 2- (3,4-dihydroxy) ethylamine of the General formula
R

CH ₂ CH ₂ NHR ² where R ¹ = R ² C (O) C ₆ H ₄ NO ₂ , -C (O) CH (CH ₃ ) ₂ , -C (O) C ₆ H ₁₁ , -C (O) CH ₂ C ₆ H ₁₁ , -C (O) CH ₂ C ₅ H ₉ , -C (O) (CH ₂ ) ₄ Cl, -C (O) (CH ₂ ) ₃ Cl, -C (O) CH ₂ Cl, -C (O) -CH (C ₃ H ₇ ) ₂ , -SO ₂ C ₆ H ₄ CH ₃ , -SO ₂ C ₆ H ₄ Cl,

or R ¹ = H, and R ² takes the indicated values.

 These compounds are prepared in a known manner by the Einhorn method by reacting dopamine hydrochloride with acid chlorides in pyridine. Elemental analysis data, melting points, yields of synthesized compounds, and IR spectral parameters are presented in Table 1.

 Example 1 Preparation of N- {2- [3,4-bis- (4-nitrobenzoyloxy) phenyl] ethyl} -4-nitrobenzamide (compound 1).

In a three-necked flask equipped with a stirrer, dropping funnel and reflux condenser, load 3 g of dopamine and 40 ml of pyridine. The reaction mixture was cooled in an ice bath to a temperature of 5 ± 2 ° ^C. Then, while stirring, 9 g of acid chloride of 4-nitrobenzoic acid. The reaction mixture was stirred at 5-10 ^C. for 2 hours. The pyridine hydrochloride formed was filtered off, the mother liquor was poured into chilled water (≈ 100 ml). The reaction mixture was stirred at room temperature for 2-3 hours, the precipitated product was crystallized for 20 hours. The precipitate of N- {2- [3,4-bis- (4-nitrobenzoyloxy) phenyl] ethyl} -4-nitrobenzamide was filtered off, washed with acidified water. and alcohol, dried in air to constant weight. Obtain 7.5 g of a white to yellowish crystalline powder. Mp 205-208 ° C. The product yield is 79.1% of the dopamine hydrochloride taken in the reaction. Purification of the product is carried out by recrystallization from dimethylformamide or from acetone. The structure of the compound is confirmed by elemental analysis, methods of IR and PMR spectroscopy (see table 1).

 Compounds 2-12 were similarly prepared.

 Example 2 Preparation of N- {2- [4-chlorobutylcarboxy) -3-hydroxyphenyl] ethyl} -4-chlorobutylcarboxamide (compound 13).

In a three-necked flask equipped with a stirrer, dropping funnel and reflux condenser, load 3 g of dopamine and 40 ml of pyridine. The reaction mass is cooled to a temperature of 5 ± 2 ^about CM. Then, 5.0 g of ω-chlorovaleric acid chloride is added with stirring. The reaction mixture was stirred at 5-10 ^C. for 2 hours. The pyridine hydrochloride was filtered off, the mother liquor was poured into chilled water (≈ 100 ml) and stirred at room temperature for 3 hours. Water was decanted and the remaining oily residue was dried in vacuo to an oil pump to constant weight. The product is purified on a chromatographic column (d 2 cm) with silica gel. Eluent chloroform. The composition of the fraction is monitored by TLC on Silufol UV-254 plates. Combine fractions having the same composition. The solvent is distilled off. Obtain 0.8 g of compound 7 in the form of a slightly yellowish oil and 1.9 g of compound 13 in the form of crystalline white powders with a creamy tint. The yield of products is 10.0 and 30.3%, respectively (see table 1).

Using the PMR method, the structure of newly synthesized triacyl (R ₁ = R ₂ = acyl; compound 1-12) with diacyl (R ₁ = H, R ₂ = acyl; compound 13 of 14) derivatives of dopamine was confirmed. PMR spectra were recorded in DMSO-d ₆ on a Tesla NMR spectrometer BS-587A (80 MHz); TMS internal standard; the sample temperature is 303 K. The following absorption signals are recorded in the PMR spectra of dopamine hydrochloride: a wide absorption signal with an intensity of two proton units at δ 8.82 ppm belonging to protons of hydroxyl groups; wide proton absorption signal at a quaternary nitrogen atom with an intensity of three proton units at δ 8.02 ppm absorption signals of aromatic protons: H-5, ∂δ 6.69 ppm I _5.6 = 7.9 Hz; H-2, ∂ δ 6.63 ppm I _2.6 2.0 Hz; H-6, ∂∂ δ 6.47 ppm I _5.6 7.9 Hz.

 In the region of δ 2.5-3.0 ppm unresolved multiplet of absorption signals of four aliphatic protons are recorded. As an example, we consider the spectra of dopamine derivatives and chlorvaleric acid (compounds 7 and 13).

In the 1H-NMR spectra of all acyl derivatives of dopamine, the absorption signals of aliphatic protons of the acyl part of the molecule are complex unresolved multiplets in the region δ = 1.2-3.5 ppm. also overlapping with the absorption signals of the DMSO solvent and the absorption signal of H ₂ O (impurity in DMSO). Therefore, taking into account the data of the PMR spectrum of dopamine hydrochloride, the most informative region of the PMR spectra obtained by dopamine derivatives is the low-field part of these spectra. In the low-field part of the spectra of triacyl derivatives, a triplet of the absorption signal of the amide proton was recorded at δ 7.89 ppm. and I _NH1CH2 5.4 Hz and an unresolved multiplet of three aromatic protons at δ7.00-7.25 ppm. These spectra are characterized by the absence of absorption signals of hydroxyl protons. In the low-field part of the 1H-NMR spectra of diacyl derivatives of dopamine, the absorption signal of the hydroxyl proton was recorded at δ 9.47 ppm. amide proton absorption triplet at δ 7.85 ppm and I _NH1CH2 5.5 Hz, as well as absorption signals of aromatic protons:
H-5, ∂δ 6.88 ppm I _5.6 8.1 Hz;
H-2, ∂∂ δ 6.78 ppm I _2.6 1.8 Hz;
H-6, ∂∂δ 6.60 ppm I _6.2 1.8 Hz, I _5.6 8.1 Hz.

 Thus, using the PMR method, the presence of three and two acyls in the molecule of dopamine derivatives was confirmed.

 The presence of the proton absorption signal of one of the hydroxyl groups in the PMR spectrum of the diacyl derivative of dopamine allows the position of the free hydroxyl group in the aromatic ring of the molecule to be determined using special techniques of PMR spectroscopy.

When suppressing the spin-spin interaction of β-methylene protons of a dopamine molecule with protons of the aromatic ring at the absorption frequency of β-methylene protons, a narrowing of the absorption signals of H-2 and H-6 protons is observed. The position of the hydroxyl group of the diacyl derivative is established on the basis of changes in the intensity of the absorption signals of aromatic protons when applying the homonuclear Overhauser effect (NEA). A significant increase in the intensity of the H-2 proton absorption signal was detected in the experiments on nuclear-electronic investigations when the proton signal of the hydroxyl group was saturated. This indicates the spatial proximity of the latter to the hydroxyl group of the diacyl derivative, unequivocally proving that the hydroxyl group is in the m-position to the —CH ₂ CH ₂ NHR ² group.

Immunotropic activity was evaluated by the ability of the drugs to change:
the formation of antibodies to test antigens (sheep erythrocytes);
delayed-type hypersensitivity reaction;
graft versus host reaction;
phagocytic activity of neutrophils;
bone marrow cell proliferation;
infection resistance of experimental animals,
In the experiments, male mice of hybrids (СВАхС57В1) F1, and nonlinear mice weighing 20–25 g were used. Animals were kept at a temperature of 20–21 ° C at 12 h light conditions, access to feed ad libitum.

The studied preparations were administered intraperitoneally or subcutaneously in the form of a suspension in 0.1 ml of physiological saline containing 0.1% Tween 80 (0.17% Tween 80 was used in separate experiments). The control animals were injected with an equal volume of physiological saline containing Tween 80. Injections were carried out according to the scheme of 0.1, 2 and -1, 0.1, where 0 day of immunization. To induce antibody formation, mice were immunized with a erythrocyte drum at a dose of 107 cells per mouse. After 6 days of immunization, mice were sacrificed and hemagglutimin titers and serum hemolytic activity were determined spectrophotometrically (A. A. Burkin, A. S. Losev Chem. Pharm. Journal. 1976, N 11, pp. 41-45) in micromodification. When using the delayed-type hypersensitivity reaction (HRT), mice were sensitized intraperitoneally with Staphylococcus albumen cells at a dose of 5 × 10 ⁶ cells per mouse. After 5 days, a resolving dose of Staphylococcus albumen 10 ⁷ cells in 0.05 ml was injected into the paw pad of the hind paw, after 24 hours the difference in the masses of the control and inflamed paws was measured.

In separate experiments, ram erythrocytes were used as a test antigen, a sensitizing dose of 3 × 10 ⁷ , a resolving dose of 10 ⁸ cells.

When determining the effect of the studied compounds on the phagocytic activity of neutrophils, 24 hours after intraperitoneal injection of drugs, blood was taken into a heparin solution (final concentration of 10 units / ml). Then, 0.1 ml of blood was mixed with 0.05 ml of a suspension of Staphylococcus albumen cells, incubated for 30 min at ³⁷ ° C, red blood cells lysed using 0,83% NH ₄ Cl, made fixed smears and alcohol. After Ramonovsky-Giemsa staining, the number of phagocytic cells from 200 neutrophils (phagocytosis activity) and the average number of microbial cells absorbed by one neutrophil (phogocytic index) were calculated. In assessing the activity of neutrophil superoxide production of blood obtained under these conditions, mixed 1: 1 with a 0.2% solution of nitro blue tetrazolium (NBT), incubated 30 min at ³⁷ ° C and smears made. The smears were stained with safranin and the number of neutrophils containing granules of reduced nitrotetrazolium from 200 cells was counted.

In the study of the effect on bone marrow cell proliferation, mice were sacrificed by cervical dislocation 24 hours after a single intraperitoneal injection of drugs. Then, under the sterile conditions, the tibia was removed, from which bone marrow cells were washed with 199 medium. After centrifugation and washing, the cells were cultured in 96-well plates for immunological reactions for 16 hours at ³⁷ ° C in a humidified atmosphere of 5% CO _2. 10% fetal calf serum and ³ N-thymidine (1 μm per well) were added to the incubation medium. After the incubation time, the cells were transferred to FN-8 paper filters. The filters were dried, washed 2 times for 5 min with saline, followed by 5% TCA at 4 to 24 ^{° C} and 2 x 5 minutes 5% TCA. After drying, the radioactivity of the samples in the ZhS-8 scintillator was calculated on a counter.

The graft versus host reaction was determined in the popliteal lymph nodes with local administration of semi-allogeneic cells. To mice, hybrids (СВАхС57В1) FI, 2 × 10 ⁷ cells isolated from the lymph nodes (inguinal, popliteal, axillary, cervical) of the parental genotype of the CBA line were introduced into the pillow of the hind paw. The same number of syngeneic cells from lymph nodes was injected into the contralateral paw. The animals of the experimental group were injected with the studied substances for three days, starting 24 hours before the transfer of lymphocytes. On the eighth day, the mice were sacrificed by cervical dislocation, subcolumn lymph nodes were removed into the Hanks solution, and their mass was determined.

При изучении влияния препарата на резистентность мышей к инфекции использовали суточную культуру Salmonella enteritidis. Животных заражали подкожной инъекцией 5˙10⁸ клеток на мышь. Исследуемые препараты вводили в течение 4 дней, начиная за сутки до заражения. Эффект препарата оценивали по средней продолжительности жизни животных в группе. При определении острой токсичности подсчитывали количество погибших мышей после однократной внутрибрюшинной инъекции суспензии исследуемых веществ. В качестве вещества сравнения был выбран хорошо известный иммуномодулятор левамизол. Необходимо отметить, что данные о его фармакологической активности противоречивы, в зависимости от иммунного статуса, дозы и схемы введения левамизол стимулирует или угнетает иммунитет (B. Renoux, Drugs,

1980, т.19, с. 89-99). Однако сочли возможным использовать его в качестве вещества сравнения, так как иммуномодуляторы, близкие по структуре и типу действия, отсутствуют и, кроме того, это наиболее изученный препарат. В связи с тем, что левамизол не влиял на пролиферацию клеток костного мозга, в этом эксперименте в качестве вещества сравнения использовали известный препарат метилурацилстимулятор лейкопоэза. При изучении влияния препаратов на резистентность мышей к инфекциям для сравнения использовали препарат "Бронхомунал" (ЛЕК, Югославия), применяемый для лечения бронхолегочных инфекций.The reaction was evaluated by the reaction index-IR:
IR

When studying the effect of the drug on the resistance of mice to infection, the daily culture of Salmonella enteritidis was used. Animals were infected by subcutaneous injection of 5 × 10 ⁸ cells per mouse. The studied drugs were administered for 4 days, starting a day before infection. The effect of the drug was evaluated by the average life expectancy of the animals in the group. When determining acute toxicity, the number of dead mice was calculated after a single intraperitoneal injection of a suspension of the test substances. The well-known immunomodulator levamisole was chosen as the reference substance. It should be noted that data on its pharmacological activity are contradictory, depending on the immune status, dose and administration schedule, levamisole stimulates or inhibits immunity (B. Renoux, Drugs,

1980, t. 19, p. 89-99). However, it was considered possible to use it as a reference substance, since immunomodulators that are similar in structure and type of action are absent and, in addition, it is the most studied drug. Due to the fact that levamisole did not affect the proliferation of bone marrow cells, in this experiment, the well-known preparation methyluracil stimulator of leukopoiesis was used as a comparison substance. When studying the effect of drugs on the resistance of mice to infections, Bronchomunal (LEK, Yugoslavia) used to treat bronchopulmonary infections was used for comparison.

 The results of studies of the immunotropic activity of the claimed compounds are presented in table.2-5.

 It was found that derivatives of 2- (3,4-dioxiphenyl) ethylamine are able to change the activity of the immune system (Tables 2, 3). The severity and direction of the effect depend on the substituent. Most of the tested substances stimulated immunological reactions, while compound 14 depressed cellular immunity. Of the compounds studied, the highest immunostimulating activity was observed in compounds 1 and 2. Both substances strongly stimulate the formation of antibodies to the test antigen (Table 2), and drug 1 reacts to some extent with HRT (table 2). In addition, these substances normalize the reduced graft versus host reaction and bone marrow cell proliferation (Table 4.5).

 The study of new derivatives of 2- (3,4-dihydroxyphenyl) ethylamine on the ability to inhibit the replication of the virus was carried out as follows.

CEM-SS cells were grown in RPMI 1640 medium containing 10% inactivated fetal bovine serum, 2 μM glutamine and 50 μg / ml gentamicin. The HIV-1 BRU isolate was propagated by infection of CEM-SS cells. Starting from day 3 of infection, the medium was collected daily and filtered on CEM-SS cells. Strains were stored in portions at -80 ° ^C. Reproduction of HIV-1 BRU in CEM-SS cells was measured by counting sintsitsy, produced 4 days post infection. CEM-SS cells were infected with 100-200 syncytia of HIV-forming units per 400,000 cells. After 30 minutes of absorption, residual free virus was separated. The infected cells were resuspended in RPMI medium containing 10% fetal bovine serum and 0.9 ml (400,000 cells) were added to the wells of 24-well plates. Then added 0.1 ml of various dilutions of antiviral drugs, and cultured at 37 ° ^C. After 4 days sintsitsy were counted by light microscopy. Inhibition of virus propagation was confirmed by comparing the activity of reverse transcriptase associated with a particle of the virus isolated 4 days after infection in the presence or absence of the drug. Cytotoxicity was determined by measuring the decrease in viability of uninfected cells in the presence of the drug using the MTT repair method.

 When testing antiviral activity used a series of increasing concentrations of drugs. The value given in Table 6 corresponds to the maximum concentration of the test substance used in the experiment. The activity of the drug was expressed as the minimum concentration causing inhibition of virus replication. Azidothymidine was used as the reference substance.

 As can be seen from the data in table 6, azidothymidine inhibits the replication of viruses at a concentration of 0.01 μm, while compound 2 at a concentration of 0.1 μm. However, azidothymidine is much more toxic: at a concentration of 0.1 μM, death of 20% of cells is observed, and compound 2 even at a concentration of 10 μM does not show toxicity. Therefore, given both of these factors (activity and toxicity), the effects of the claimed compound 2 and azidothymidine should be considered comparable. Studies of 2- (3,4-dihydroxyphenyl) ethylamine derivatives for anti-HIV-1 analysis indicate that compound 2 can inhibit virus replication.

 As a result of the above investigations, it was found that 2- (3,4-dihydroxyphenyl) ethylamine derivatives are significantly less toxic than the levamisole preparation. In terms of immunomodulatory activity, they are not inferior to comparison drugs, and in some respects surpass them. The spectrum of immunotropic activity of these compounds differs from the spectrum of activity of levamisole. Levamisole, as you know, mainly activates cellular reactions, while, for example, compounds 1 and 2 more pronouncedly stimulate humoral reactions.

 These circumstances allow us to recommend derivatives of 2- (3,4-dihydroxyphenyl) ethylamine as potential immunostimulating drugs for the treatment of diseases associated with impaired immune system function, and also as a means to reduce the side immunosuppressive effects of other drugs, for example cytostatics, and as antiviral agents.

Claims (2)

1. Derivatives of 2- (3,4-dihydroxyphenyl) ethylamine of the general formula

where R ₁ R ₂ (C (O) C ₆ H ₄ NO ₂ , C (O) CH (CH ₃ ) ₂ , C (O) CH / (C ₃ H ₇ ) ₂ , C (O) C ₆ H _{1 1} , C (O) CH ₂ C ₆ H _{1 1} , C (O) CH ₂ C ₅ H ₉ , C (O) CH ₂ Cl, C (O) (CH ₂ ) ₃ Cl, C (O) (CH ₂ ) ₄ Cl, SO ₂ C ₆ H ₄ CH ₃ , SO ₂ C ₆ H ₄ Cl,

or R ₁ H, and R ₂ has the indicated meanings,
showing immunotropic activity.  2. N- {2 [3,4-Bis (isobutyryloxy) phenyl] ethyl} isobutyrylamide, which has the ability to inhibit the replication of the virus.

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