RU2794763C1 - 1-hydroxy- and 1-methoxy-2-(4-nitrophenyl)imidazole derivatives with antiviral activity against orthopoxviruses - Google Patents

Abstract

FIELD: fine organic synthesis; medicine; virology.

SUBSTANCE: invention relates to the field of organic chemistry, specifically to new derivatives of 1-hydroxy- and 1-methoxy-2-(4-nitrophenyl)imidazole of general formula Ia-derivatives.

EFFECT: compounds of formula Ia-d R = H; R¹ = R² = CH₃ (Ia); R=H; R¹ R² = CH₂C(CH₃)₂CH₂ (Ib); R = R¹ = R² = CH₃ (Ic); R= CH₃; R¹ R² = CH₂C(CH₃)₂CH₂ (Id) have antiviral activity against orthopoxviruses.

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Description

The invention relates to the field of fine organic synthesis, medicine and virology, and in particular to the use of organic biologically active compounds of General formula Ia-d (figure 1) as inhibitors of orthopoxviruses and promising chemical compounds in the creation of drugs for the treatment and emergency prevention of orthopoxvirus diseases in humans and animals.

Since 1980, universal vaccination of the population against smallpox, the causative agent of which is the variola virus (VAV), has been discontinued in connection with the announcement by the World Health Organization (WHO) of the complete eradication of this disease on the planet. Therefore, at present, more than half of the world's population is deprived of anti-small immunity. Traditional vaccination using live vaccinia virus is dangerous for people with immunodeficiency, whose number is increasing every year [UNAIDS date 2017; Pelkonen P.M, Tarvainen K., Hynninen A., Kallio E.R., Henttonen K., Palva A., Vaheri A., Vapalahti O. Cowpox with severe generalized eruption, Finland. Emerg. Infect. Dis. 2003. 9:1458-1461]. It should be noted that in the Russian Federation there are no officially registered chemotherapy drugs for the treatment of post-vaccination complications and the prevention of smallpox diseases.

The nucleoside intravenous drug Cidofovir (Cidofovir, Vistide®, CDV) selectively inhibits viral DNA polymerase and exhibits anti-orthopoxvirus activity in in vitro and in vivo experiments [ J. Gen. Virol . 2016. 97(5):1229-1239]. Shows activity in lethal models of orthopoxvirus infection in mice and monkeys. However, significant disadvantages of cidofovir are low oral bioavailability and potential nephrotoxicity. In addition, this drug was ineffective when used after the onset of smallpox lesions in BHO-infected monkeys [WHO Identification Number: WHO/HSE/GAR/BDP/2010.3/RUS. World Health Organization. Scientific review of variola virus research, 1999-2010. 2011. 132 p.].

A modification of the chemotherapy drug Cidofovir (Cidofovir) is a modern, effective and bioavailable oral antiviral chemical drug Brincidofovir (Brincidofovir, CMX001 [Chimerix Receives US Food and Drug Administration Approval for TEMBEXA® (brincidofovir) for the Treatment of Smallpox (URL: https:// www.globenewswire.com/news-release/2021/06/04/2242292/25619/en/Chimerix-Receives-US-Food-and-Drug-Administration-Approval-for-TEMBEXA-brincidofovir-for-the-Treatment- of-Smallpox.html, 04.06.2021 ]. Smallpox Research Program Review (AGIES) Notes on the 1999-2010 Variola Virus Scientific Review 2010. 43 pp.].

Also, a drug approved for the treatment of smallpox and monkeypox is Tecovirimat (Tecovirimat, containing as an active pharmaceutical substance (APS) N - {3,5-Dioxo-4-azatetracyclo [5.3.2.0 ^{2.6} .0 ^{{ 8,10}} ]dodec-11-en-4-yl}-4-(trifluoromethyl)benzamide (ST-246) [FDA approves the first drug with an indication for treatment of smallpox. FDA News Release. https://www .fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm613496.htm; 07/13/2018]. a p37 protein present in all orthopoxviruses.The disadvantage of this drug is the development of resistance of orthopoxviruses with its long-term use [WHO Identification number: WHO/HSE/GAR/BDP/2010.4. World Health Organization. WHO Advisory Group of Independent Experts to Review the Smallpox Research Program ( AGIES). Comments on the Scientific Review of Variola Virus Research 1999-2010 2010. 43 p.].

Recently, phase 1 clinical trials of a new domestic drug NIOH-14, which is a prodrug of ST-246, and by the mechanism of action, as well as ST-246, is an inhibitor of the p37 viral protein, have been completed. NIOCH-14 exhibits high antiviral activity against orthopoxviruses, including VNO [Mazurkov O.Yu., Shishkina L.N., Bormotov N.I., Skarnovich M.O., Mazurkova N.A., Chernonosov A.A. , Tikhonov A.Ya., Selivanov B.A. Evaluation of the absolute bioavailability of the chemical substance of the anti-small preparation NIOCH-14 in experiments on mice. Bulletin of Experimental Biology and Medicine . 2020. V. 170, No. 8. - S. 173-177. Doi: http://iramn.ru/journals/bbm/2020/8/5903/].

WHO experts recommend continuing research on the creation of new drugs with activity against orthopoxviruses. New antivirals may target different stages of the viral cycle [WHO ID: WHO/HSE/GAR/BDP/2010.4. World Health Organization. WHO Independent Expert Advisory Group for the Review of the Smallpox Research Program (AGIES). Comments on the Scientific Review of Variola Virus Research 1999-2010 2010. 43 p.].

In this regard, the development of new anti-small preparations with high antiviral activity and low cytotoxicity is an important task for ensuring the safety of human health and life.

The technical objective of the present invention is the synthesis of a new class of inhibitors with a high selectivity index for orthopoxviruses.

The technical result is the expansion of a number of compounds with high antivirulent activity against orthopoxviruses.

The problem is solved by synthesizing new compounds of General formula Ia-d (figure 1), with a high selectivity index for orthopoxviruses.

The synthesis of 1-hydroxy- and 1-methoxy-2-(4-nitrophenyl)imidazole derivatives Ia-d is carried out by reacting the corresponding oximes with nitrobenzaldehyde and ammonium acetate in glacial acetic acid at room temperature to obtain 1-hydroxy-2-(4- nitrophenyl)imidazoles Ia-Ib and subsequent methylation at room temperature in the presence of potassium hydroxide in DMF to obtain 1-methoxy-2-(4-nitrophenyl)imidazoles Ic-d (figure 2).

The cytotoxicity and antiviral activity of the synthesized derivatives against vaccinia, cowpox and mousepox viruses are evaluated using an adapted colorimetric method in Vero cell culture [Selivanov B.A., Tikhonov A.Ya., Belanov E.F., Bormotov N.I. , etc. Chemical and Pharmaceutical Journal. (2017) 51(6), 13-17]. The commercially available drug Cidofovir (HeritageConsumerProducts, LLC, USA) is used as a positive control.

In the prior art there is no information about the claimed compounds with high antiviral activity and low cytotoxicity.

The invention is confirmed by the following examples:

Example 1 1-(1-Hydroxy-4-methyl-2-(4-nitrophenyl)-1H-imidazol-5-yl)ethanone Ia.

Mixture 4.53 g (30.0 mmol) 4-nitrobenzaldehyde, 3.87 g (30.0 mmol) of 3-hydroxyamino-2,4-pentanedione and 2.53 g (32.5 mmol) of ammonium acetate in 25 ml of glacial acetic acid are stirred at room temperature for 12 hours and left for two days. The precipitate formed is filtered off and washed with 15 ml of diethyl ether and boiled in 200 ml of n-hexane. 5.06 g (65%) of 1-(1-hydroxy-4-methyl-2-(4-nitrophenyl)-1H-imidazol-5-yl)ethanone (Ia) are obtained in the form of a yellow powder with so pl. 182-184°C.

¹ H NMR (300 MHz, CDCl ₃ , 298 K), δ (ppm): 14.14 (s, 1H, OH); 8.44 (d, J=9.2 Hz , 2H, H-Ar); 8.32 (d, J=9.1 Hz , 2H, H-Ar); 2.63 (s, 6H, 2CH ₃ )

¹ H NMR (400 MHz, DMSO-d ₆ , 295 K), δ (ppm): 12.63 (br. s, 1H, OH); 8.40-8.34 (m, 4H, H-Ar); 2.55 (s, 3H, CH ₃ ); 2.42 (s, 3H, CH ₃ ) ppm

¹³ C NMR (100.62 MHz, DMSO-d ₆ ), δ (ppm): 188.5, 147.6, 134.0, 128.5, 127.9, 124.4, 124.3 , 30.3, 16.3.

IR (KBr, ν, cm- ¹ ): 2300-2800 (NH…O=C); 1648 (C=O); 1513, 1339 (NO ₂ ).

Example 2 3-hydroxy-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrohydro-4H-benzimidazol-4-one Ib.

A mixture of 4.53 g (30 mmol) 4-nitrobenzaldehyde, 5.07 g (30 mmol) 2-(hydroxyimino)-5,5-dimethylcyclohexane-1,3-dione and 2.69 g (35 mmol) ammonium acetate in 30 ml of glacial acetic acid is stirred at room temperature for 6 hours. The precipitate formed is filtered off and washed with 20 ml of water. 6.26 g (69%) of 3-hydroxy-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrohydro-4H-benzimidazol-4-one (Ib) are obtained in powder form. yellow color. So pl. 130-132°C.

¹ H NMR (400 MHz, DMSO-d ₆ , 295 K), δ (ppm): 2.81 (br. s, 1H, OH); 8.34 (s, 4H, H-Ar); 2.68 (s, 2H, CH ₂ ); 2.39 (s, 2H, CH ₂ ); 1.06 (s, 6H, 2CH ₃ ).

¹³ C NMR (100.62 MHz, DMSO-d ₆ ), δ (ppm): 186.8, 172.5, 150.4, 147.9, 142.6, 134.3, 129.0 , 124.4, 124.3, 53.0, 38.8, 35.8, 28.4, 21.5.

IR (KBr, ν, cm ^-1 ): 2300-2800 (N-OH...O=C); 1666 (C=O); 1520, 1342 (NO ₂ ).

Example 3 1-(1-Methoxy-4-methyl-2-(4-nitrophenyl)-1H-imidazol-5-yl)ethanone Ic.

A mixture of 2.00 g (8 mmol) 1-(1-hydroxy-4-methyl-2-(4-nitrophenyl)-1H-imidazol-5-yl)ethanone Ia, 0.50 g (9 mmol) potassium hydroxide in 20 ml of DMF are cooled with stirring to 4° C. and 0.5 ml (1.14 g, 8 mol) of methyl iodide are added. The reaction mass is kept for 4 hours at room temperature, then poured into 60 ml of water, stirred for 20 minutes. The precipitate formed is filtered off and washed with water on the filter. Then boil in 22 ml of n-hexane. 1.56 g (71%) of 1-(1-methoxy-4-methyl-2-(4-nitrophenyl)-1H-imidazol-5-yl)ethanone (Ic) is obtained in the form of a bright yellow powder. So pl. 122-124°C.

¹ H NMR (300 MHz, CDCl ₃ , 303 K), δ (ppm): 8.36 (d, J=9.3 Hz, 2H, H-Ar); 8.32 (d, J=9.1 Hz, 2H, H-Ar); 3.97 (s, 3H, OCH ₃ ); 2.60 (s, 3H, CH ₃ ); 2.59 (s, 3H, CH ₃ ).

¹ H NMR (400 MHz, DMSO-d ₆ , 294 K), δ (ppm): 8.37 (d, J=9.1 Hz, 1H, H-Ar); 8.30 (d, J=9.0 Hz , 1H, H-Ar); 3.96 (s, 3H, OCH ₃ ); 3.55 (s, 3H, CH ₃ ); 2.47 (s, 3H, CH ₃ ).

¹³ C NMR (150.94 MHz, DMSO-d ₆ ), δ (ppm): 187.3, 148.1, 143.8, 139.5, 133.5, 128.9, 125.8 , 124.7, 67.9, 30.2, 17.1.

IR (KBr, ν, cm ^-1 ): 3116, 3010, 2925 (2CH ₃ ; OCH ₃ ); 1657 (C=O); 1511, 1338 (NO ₂ ).

Example 4 3-methoxy-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrahydro-4H-benzimidazol-4-one Id.

A mixture of 2.1 g (7 mmol) 3-hydroxy-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrohydro-4 H -benzimidazol-4-one Ib, 0.56 g (10 mmol) of potassium hydroxide in 20 ml of DMF is cooled with stirring to 4°and add 0.4 ml (0.99 g, 7 mmol) of methyl iodide. The reaction mass is kept for 4 hours at room temperature and left for two days. Then poured into 100 ml of water, stirred for 20 minutes. The precipitate formed is filtered off and washed with water on the filter. Then boiled in 20 ml of water, filtered while hot. 1.60 g (73%) of 3-methoxy-6,6-dimethyl-2-(4-nitrophenyl)-3,5,6,7-tetrahydro-4H-benzimidazol-4-one (Id) is obtained as a powder bright yellow. So pl. 148-150°C.

¹ H NMR (400 MHz, DMSO-d ₆ , 291 K), δ (ppm): 8.39-8.32 (m, 4H, H-Ar); 4.08 (s, 3H, OCH ₃ ); 2.72 (s, 2H, CH ₂ ); 2.45 (s, 2H, CH ₂ ); 1.08 (s, 6H, 2CH ₃ ).

¹³ C NMR (100.62 MHz, DMSO-d ₆ ), δ (ppm): 186.5, 151.2, 148.3, 141.9, 133.5, 129.0, 124.6 , 122.7, 67.7, 52.7, 38.8, 35.8, 28.4.

IR (KBr, ν, cm ^-1 ): 3086, 2963, 2893 (2CH ₃ ; OCH ₃ ); 1666 (C=O); 1520, 1342 (NO ₂ ).

Spectral analysis of the compounds obtained was carried out on a Bruker Avance TM 400 spectrometer (operating frequency ¹ H: 400 MHz, ¹³ C: 100.62 MHz) using deuterated solvents (CDCl ₃ , DMSO-d6). Residual solvent signals were used as internal standards. The IR spectrum was recorded on a Shimadzu IRAffinity-1 FTIR Fourier spectrophotometer in KBr pellets.

Melting points were measured on a PTP(M) melting point apparatus and are uncorrected.

Example 5. Determination of cytotoxicity and antiviral activity of compounds Ia-d against vaccinia viruses (Copenhagen strain), cow pox (Grishak strain) and mouse pox (K-1 strain) in Vero cell culture

The cytotoxicity and antiviral activity of the synthesized derivatives against vaccinia, cowpox and mousepox viruses are evaluated using an adapted colorimetric method in Vero cell culture [Selivanov B.A., Tikhonov A.Ya., Belanov E.F., Bormotov N.I. , etc. Chemical and Pharmaceutical Journal. (2017) 51(6), 13-17]. The commercially available drug Cidofovir (HeritageConsumerProducts, LLC, USA) is used as a positive control.

The antiviral activity and cytotoxicity of the claimed compounds Ia-d are tested in Vero cell culture. As a model, vaccinia (Copenhagen strain), cowpox (Grishak strain) and mouse pox - ectromelia (K-1 strain) viruses are used as typical representatives of the orthopoxvirus family from the State Collection of Causative Agents of Viral Infections and Rickettsiosis FBSI SRC BV "Vector" of Rospotrebnadzor.

A monolayer of Vero cells is grown in 96-well plates. Solutions in dimethyl sulfoxide with a concentration of 20 mg/ml are prepared from the studied compounds Ia-d. From the prepared solutions, a series of dilutions in a nutrient medium is prepared with a 5-fold or 3-fold step, the initial concentration of the drug in the well of the tablet is 100 or 400 μg/ml.

The dilutions of the preparations are added to the wells of the plates with a monolayer of cells, then the virus is introduced into half of the wells of the plate, and the second half of the wells of the plate is used to assess the cytotoxicity of the compounds.

As a reference drug (or prototype) use the drug Cidofovir (Cidofovir manufactured by Gilead Sciences Inc., USA).

After incubation for 4 days, the vital dye neutral red is added to the wells, which is absorbed only by living cells. Cells that are destroyed by the virus or damaged by the toxic effects of the compound do not take up the vital dye. The monolayer of cells is washed from the unabsorbed dye with physiological saline, and a lysis buffer is introduced into the wells, which destroys cells with absorbed dye and dissolves this dye.

Light absorption (optical density - OD) solutions in the wells of the tablets is measured at a wavelength of 490 nm on a tablet spectrophotometer E-Max (Molecular Devices USA). OD is proportional to the number of viable cells, it reflects the antiviral activity of the compound in comparison with the OD in the virus control or characterizes the cytotoxicity of this compound in comparison with the OD in the cell control.

The results are processed using the Soft Max Pro 4.0 program, which calculates the 50% cytotoxic concentration (CC ₅₀ μg/ml) and the 50% virus inhibitory concentration (IC ₅₀ μg/ml). Based on the indicators of SS ₅₀ and IC ₅₀ determine the selectivity index (SI)

SI = CC ₅₀ / IC ₅₀ .

An SI value of less than 8 is considered unacceptable for compounds that may be promising as antivirals [ J. Gen. Virol . 2016. 97(5):1229-1239].

Calculation and comparison of the average values of CC ₅₀ and IC ₅₀ carried out by standard methods of descriptive statistics using the data analysis package Microsoft Excel.

The results of studying the cytotoxic and antiviral effects of the compounds are shown in Table 1.

Table 1. Indicators of cytotoxicity and antiviral activity of chemical compounds Ia-d against vaccinia, cowpox and ectromelia viruses in Vero cell culture. Preparations
(mm) CC ₅₀ , M±Sm, n=3,
µg/ml (µmol) IC ₅₀ , M±Sm, n=3, μg/ml (μmol)
(SI=CC ₅₀ /IC ₅₀ ) vaccinia virus Virus
cowpox ectromelia virus Ia
(261.24) 7.40±1.83*
(28.48±7.01) 0.17±0.05**
(0.65±0.19)
(43.5) 0.64±0.21**
(2.45±0.80)
(11.6) 0.34±0.10**
(1.30±0.38)
(21.8) Ib
(301.30) 42.91±14.56
(142.42±48.32) 0.04±0.01**
(0.13±0.03)
(1072.8) 0.35±0.11**
(1.16±0.36)
(122.6) 0.12±0.04**
(0.40±0.13)
(357.6) ic
(275.26) 122.10±41.34
(443.6±150.2) 1.31±0.03**
(4.76±0.11)
(93.2) 4.38±1.57**
(15.91±5.70)
(27.9) 3.82±0.31 ^&
(13.88±1.13)
(32.0) ID
(315.33) 364.00±85.85
(1154.3±272.2) 1.25±0.07**
(3.96±0.22)
(291.2) 5.65±1.54**
(17.92±4.88)
(64.4) 3.95±0.18 ^&
(12.53±0.57)
(92.2) Cidofovir
(279.19)
(prototype) 310.33±60.09
(1111.5±215.2) 13.02±4.19
(47.14±15.01)
(23.8) 25.56±4.45
(91.55±15.94)
(12.1) 6.18±2.62
(22.14±9.38)
(50.22)

Note:

CC ₅₀ - 50% cytotoxic concentration of drugs, at which 50% of cells in an uninfected monolayer are destroyed.

IC ₅₀ - 50% virus inhibitory drug concentration that retains 50% of the cells in the infected monolayer.

The values of CC ₅₀ and IC ₅₀ are presented as M±Sm, where M is the mean value, Sm is the standard deviation when the number of measurements n=3.

SI - drug selectivity index (CC ₅₀ /IC ₅₀ ).

Mm is the molecular weight.

* - difference from the indicators of CC ₅₀ of all other drugs at p≤0.05.

** - difference from the corresponding indicators of IC ₅₀ Cidofovir at p≤0.05.

^& - tendency to differ from the IC ₅₀ of Cidofovir at p≤0.9.

As can be seen from table 1, derivatives of 1-hydroxy- and 1-methoxy-2-(4-nitrophenyl)imidazole compounds Ia-d are more active against vaccinia, cowpox and mousepox (ectromelia) than the reference drug (prototype ) Cidofovir.

Thus, the claimed series of compounds Ia-d is a new type of effective inhibitors of orthopoxviruses pathogenic for humans and animals.

Claims (3)

Derivatives of 1-hydroxy- and 1-methoxy-2-(4-nitrophenyl)imidazole of general formula Ia-d

, possessing antiviral activity against orthopoxviruses.

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