RU2814738C1 - Agent based on arglabin derivative, having selective cytotoxic action - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to the use of an arglabin derivative of formula Ia as an agent having selective cytotoxic action on duodenal adenocarcinoma HuTu 80.

EFFECT: effective cytotoxic action on duodenal adenocarcinoma HuTu 80 of arglabin derivative of formula Ia, which can find application in development of anticancer preparations of targeted action, having minimal side effects.

1 cl, 2 dwg, 7 ex

Description

The invention relates to the field of medicinal chemistry, in more detail to arglabin derivatives of the formula Ia - Ie , which have a selective cytotoxic effect in vitro .

Increasing the selectivity of the cytotoxic effect of chemotherapeutic drugs is an urgent task of modern medicinal chemistry, since its solution is extremely important for reducing the side effects associated with the use of chemotherapeutic drugs, as well as the creation of drugs for the specific prevention of cancer. Therefore, the discovery of selective cytotoxicity in the claimed arglabin derivatives of formula Ia - Ie against individual cancer cell lines, combined with low cytotoxicity against normal cell lines, can be used in the development of targeted antitumor drugs with minimal side effects.

The starting compound is arglabin [( 3aR , 4aS , 6aS , 9aS , 9bR )-1,4a-dimethyl-7-methylidene-5,6,6a,7,9a,9b-hexahydro- 3H -oxyreno [2′,3′:8,8a]azuleno[4,5- b ]furan-8( 4aH )-one] 1 :

Arglabin is used in medicine as an antitumor drug in the treatment of lung, liver, intestinal, stomach and breast cancer [Pharmaceutical compositions of arglabin and arglabin derivatives: Pat. WO9848789A1/SM Adekenov; application 04/26/1997; publ. 05.11.1998], [The method of treatment of locally propagated mammary gland cancer: patent. WO9958148A1/KJ Musulmanbekow; application 05/14/1998; publ. 11/18/1999], [Method of treatment of patients with initial liver cancer: pat. WO9964003A1/KJ Musulmanbekow; application 06/09/1998; publ. 12/16/1999]. Arglabin is a sesquiterpene lactone of the guayanolides class, contained in the plant wormwood ( Artemisia glabella ) [Adekenov SM Arglabin - A new sesquiterpene lactone from Artemisia glabella. / SM Adekenov, MN Mukhametzhanov, AD Kagarlitskii, AN Kupriyanov // Chem. Nat. Compd. - 1982, V. 18, No. 5. - P. 623-624]. The antitumor activity of arglabin is due to competitive inhibition of the enzymatic activity of farnesyltransferase, which is involved in the prenylation of Ras proteins [Shaikenov TE Arglabin-DMA, a plant derived sesquiterpene, inhibits farnesyltransferase. / TE Shaikenov, SM Adekenov, RM Williams, N. Prashad, FL Baker, TL Madden, R. Newman // Oncolog. Rep. - 2001, V. 8. - P. 173-182], [Lone SH Arglabin: from isolation to antitumor evaluation. / SH Lone, KA Bhat, MA Khuroo // Chem.-Biol. Interact. - 2015, V. 240. - P. 180-198]. Defarnesylation of Ras proteins inhibits the division and growth of tumor cells, since oncogenic forms of Ras proteins are detected in many malignant diseases.

A water-soluble synthetic derivative of arglabin is known - [(3a R ,4a S ,6a S ,7 R ,9aS,9b R )-7-[(dimethylamino)methyl]-1,4a-dimethyl-5,6,6a,7,9a ,9b-hexahydro-3 H -oxyreno[2′,3′:8,8a]azuleno[4,5- b ]furan-8(4a H )-one hydrochloride] 2 [Method for production of hydrochloride 1(10) beta-epoxy-13-dimethylamino-5,7alpha,6,11beta (h)-guaia-3(4)-en-6,12-olide, the lyophilized antitumor preparation "arglabin": patent. EP2069357B1 / SM Adekenov; application 09.19.2006; publ. 08.26.2015], accepted by the applicant as a prototype, which differs from natural arglabin in higher bioavailability and better pharmacokinetic properties, which allows the use of this derivative in chemotherapy of tumor diseases for intravenous administration.

From the studied state of the art in the field of medicinal chemistry, it was revealed that the synthetic derivative 2 , like the original arglabin 1 , has high cytotoxicity against various cancer cell lines, however, their cytotoxicity against normal cell lines is also high, i.e. These drugs do not have a selective effect.

As of the date of submission of application materials, the applicant did not identify information on arglabin derivatives from the researched state of the art in patent and scientific databases, which would simultaneously exhibit high cytotoxicity on cancer cells and low cytotoxicity on normal cells.

The technical result of the claimed technical solution is the development of a product based on the arglabin derivative of formula Ia , which has a selective cytotoxic effect, allowing to achieve a decrease in cytotoxicity towards normal cells while maintaining a high cytotoxic effect against cancer cells.

The essence of the claimed technical solution is the use of an arglabin derivative of formula Ia as an agent with a selective cytotoxic effect against duodenal adenocarcinoma HuTu 80:

The claimed technical solution is illustrated in Figure 1, Figure 2.

Figure 1 presents Table 1, which shows the IC ₅₀ values of arglabin and its derivatives in relation to cancer and normal cell lines of various origins.

Figure 2 presents Table 2, which shows the cytotoxic effects (μM) and selectivity index (SI) values of arglabin derivatives.

Next, the applicant provides a description of the claimed technical solution.

To achieve the stated technical result, it is accepted as a working hypothesis that the functionalization of natural arglabin by the Michael reaction, including the addition of a pronucleophilic reagent at the exocyclic carbon-carbon multiple bond of lactone 1 , will contribute to the emergence of selectivity, since the resulting adducts during metabolism in tumor cells could undergo retro -Michael reaction with the release of arglabin in situ , without significantly affecting the functions of normal cells.

The effective synthesis of a wide series of such adducts became possible thanks to the discovery of the high catalytic activity of tertiary phosphines for the functionalization of α-methylene-γ-butyrolactones, which include arglabin [Salin A. V. Phosphine-catalyzed Michael additions to α-methylene-γ-butyrolactones. / A. V. Salin, D. R. Islamov // Org. Biomol. Chem. - 2019, V. 17, No. 31. - P. 7293-7299.]. The synthesis of adducts was not previously possible using methods based on acid-base and metal complex catalysis due to the presence of various reactive functional groups in arglabin.

The claimed technical result is achieved by the fact that arglabin of natural origin 1 is subjected to functionalization at the exocyclic carbon-carbon multiple bond with one of the pronucleophilic reagents: diethyl malonate 3 , triethylphosphonoacetate 4 , O,O'-diethylcyanomethylphosphonate 5 , dibenzylphosphite 6 , 2-oxazolidinone 7 , uracil 8 , - using commercially available tri -butylphosphine of the formula n -Bu ₃ P as an organocatalyst, which leads to the formation of mono- or bi-addition products Ia - Ie .

The synthesis of compounds Ia - Ie is carried out in a solution of acetonitrile or N,N-dimethylformamide at 20°C for 1 hour in the presence of 10 mol.% n -Bu ₃ P. The molar ratio of the reagents depends on the structure of the pronucleophile used 3 - 8 . Methods for the synthesis and isolation of target products are presented below. The absolute configuration of the new asymmetric carbon centers in products Ia - Ie was assigned by analogy with other arglabin derivatives for which X-ray diffraction data are available [Salin AV Phosphine-catalyzed Michael additions to α-methylene-γ-butyrolactones. / AV Salin, DR Islamov // Org. Biomol. Chem. - 2019, V. 17, No. 31. - P. 7293-7299].

Next, the applicant provides examples of the implementation of the claimed technical solution.

Example 1. Synthesis of diethyl-2,2-bis(((1a S ,3a S ,4 S ,6a S ,6b R ,9a R )-1a,7-dimethyl-5-oxo-2,3,3a,4 ,5,6a,6b,9-octahydro-1a H -oxyreno[2',3':8,8a]azuleno[4,5- b ]furan-4-yl)methyl)malonate Ia.

N -Bu ₃ P (0.040 g, 0.2 mmol) was quickly added to a mixture of diethyl malonate 3 (0.32 g, 2 mmol) with arglabin 1 (0.984 g, 4 mmol) in acetonitrile (5 ml), and the reaction mixture was kept at 20°C within 1 hour. The solvent is then removed under reduced pressure, and the product is purified by column chromatography (SiO ₂ , eluent n -hexane/EtOAc=4:1, R _f = 0.39). Yield: 84% (1.09 g), colorless crystals, _mp. 195-197 °C (with decomposition), [α] _D ²⁶ = +86.7 ( c 2.00; CHCl ₃ ).

^1H NMR (400 MHz, _CDCl3 , δ ppm): 5.55 (br. s, 1H), 4.30 (kv, J = 7.1 Hz, 1H), 4.27 (kv, J = 7.2 Hz, 1H), 4.08 - 3.99 (m, 3H) overlaps with 4.07 (kv, J = 7.2 Hz, 1H), 2.84 - 2.71 (m, 4H), 2.37 - 2.30 (m, 2H), 2.18 - 2.09 (m, 8H), 1.93 - 1.83 (m, 4H) overlaps with 1.91 (br. s, 6H), 1.49 - 1.37 (m, 4H), 1.33 (s, 6H), 1.24 (t, J = 7.2 Hz, 6H). NMR ¹³ C{ ¹ H} (100.6 MHz, CDCl ₃ , δ ppm): 177.8, 170.5, 140.6, 124.8, 82.7, 72.4, 62.6, 62.0, 56.6, 53.6, 52.2, 42.5, 39.5, 33.5 , 22.7 , 22.3, 18.3, 13.9. IR ν (cm- ¹ ): 2991, 2968, 2951, 2926, 2897, 2859, 2833, 1772, 1756, 1720, 1437, 1372, 1343, 1313, 1284, 1268, 1255, 1209, 119 3, 1163, 1147, 1134, 1116, 1100, 1070, 1042, 1029, 1013, 992, 961, 924, 882, 858, 821, 807, 788, 773, 721, 682, 671, 655, 640, 595, 579, 5 51, 508, 492. Mass spectrum (HRMS-ESI): m/z calculated for C ₃₇ H ₄₉ O ₁₀ ⁺ [M+H] ⁺ : 653.3321, found: 653.3326.

Example 2. Synthesis of ethyl-2-(diethoxyphosphoryl)-3-((1a S ,3a S ,4 S ,6a S ,6b R ,9a R )-1a,7-dimethyl-5-oxo-2,3,3a ,4,5,6a,6b,9-octahydro-1a H -oxyreno[2',3':8,8a]azuleno[4,5- b ]furan-4-yl)-2-(((1a S ,3a S ,4 S ,6a S ,6b R ,9a R )-1a,7-dimethyl-5-oxo-2,3,3a,4,5,6a,6b,9-octahydro-1a H -oxyreno[ 2',3':8,8a]azuleno[4,5- b ]furan-4-yl)methyl)propanoate Ib .

N -Bu ₃ P (0.040 g, 0.2 mmol) was quickly added to a mixture of triethylphosphonoacetate 4 (0.448 g, 2 mmol) with arglabin 1 (1.476 g, 6 mmol) in acetonitrile (5 ml), and the reaction mixture was kept at 20°C within 1 hour. The solvent is then removed under reduced pressure, and the product is purified by column chromatography (SiO ₂ , eluent n -hexane/EtOAc=2:1, R _f = 0.50). Yield: 93% (1.33 g), white powder, _mp. 118-119°С, [α] _D ²² = +37.8 ( c 2.00; CHCl ₃ ).

NMR ¹ H (400 MHz, CDCl ₃ , δ ppm): 5.55 (s, 2H), 4.33 - 4.10 (m, 6H), 4.06 - 3.96 (m, 2H), 2.93 - 2.71 (m, 6H) , 2.56 (ddd, J = 17.9, 15.1, 6.2 Hz, 1H), 2.25 (td, J = 15.1, 7.5 Hz, 1H), 2.19 - 1.78 (m, 15H), 1.70 - 1.57 (m, 2H), 1.54 - 1.20 (m, 18H). NMR ¹³ C{ ¹ H} (100.6 MHz, CDCl ₃ , δ ppm): 178.6, 177.8, 170.7, 140.7, 124.8, 124.7, 82.5, 82.4, 72.5, 72.4, 63.72, 63.66, 62.6 (d , J = 4.0 Hz), 62.2, 62.0 (d, J = 7.0 Hz), 54.1, 53.6, 52.5, 52.2, 50.8 (d, J = 140.8 Hz), 42.44, 42.42, 42.33, 42.28, 39.5, 34.42, 34.40, 3 3.7 , 33.6, 32.90, 32.86, 22.72, 22.70, 22.6, 22.4, 18.4, 18.3, 16.6 (d, J = 6.0 Hz), 16.5 (d, J = 6.0 Hz), 14.0. NMR ³¹ P{ ¹ H} (162 MHz, CDCl ₃ , δ ppm): 24.7. IR ν (cm- ¹ ): 2925, 1773, 1723, 1437, 1375, 1314, 1238, 1213, 1167, 1150, 1119, 1096, 1053, 1016, 960, 858, 806, 762, 680, 66 3,649, 581, 546, 510. Mass spectrum (HRMS-ESI): m/z calculated for C ₃₈ H ₅₄ O ₁₁ P ⁺ [M+H] ⁺ : 717.3399, found: 717.3404.

Example 3. Synthesis of diethyl-(2-cyano-1,3-bis((1a S ,3a S ,4 S ,6a S ,6b R ,9a R )-1a,7-dimethyl-5-oxo-2,3 ,3a,4,5,6a,6b,9-octahydro-1a H -oxyreno[2',3':8,8a]azuleno[4,5- b ]furan-4-yl)propan-2-yl) phosphonate Ib .

To a mixture of O,O'-diethylcyanomethylphosphonate 5 (0.354 g, 2 mmol) with arglabin 1 (1.23 g, 5 mmol) in acetonitrile (5 ml), n -Bu ₃ P (0.040 g, 0.2 mmol) was quickly added and the reaction mixture kept at 20°C for 1 hour. The solvent is then removed under reduced pressure and the product is purified by column chromatography (SiO ₂ , eluent n -hexane/EtOAc=1:1, R _f = 0.43). Yield: 91% (1.22 g), white powder, _mp. 124-125°C, [α] _D ²³ = +52.0 ( c 1.01; CHCl ₃ ).

NMR ¹ H (400 MHz, CDCl ₃ , δ ppm): 5.60 - 5.53 (m, 2H), 4.41 - 4.23 (m, 4H), 4.14 (t, J = 10.0 Hz, 1H), 4.07 (t , J = 10.0 Hz, 1H), 2.95-2.67 (m, 5H), 2.67 - 2.51 (m, 2H), 2.25 - 1.67 (m, 11H) overlaps with 1.92 (br. s, 6H), 1.65 - 1.19 ( m, 4H) overlaps with 1.40 (dt, J = 7.1, 1.7 Hz, 6H), 1.34 (s, 3H), 1.33 (s, 3H). NMR ¹³ C{ ¹ H} (100.6 MHz, CDCl ₃ , δ ppm): 177.8, 177.6, 140.6, 140.3, 125.0, 124.9, 118.3 (d, J = 16.1 Hz), 83.0, 82.8, 72.4, 72.3 , 65.0 (d, J = 8.0 Hz), 64.8 (d, J = 7.0 Hz), 62.7, 62.5, 53.4, 52.1, 52.0, 51.9, 43.6 (d, J = 7.0 Hz), 43.4 (d, J = 5.0 Hz), 40.4 (d, J = 142.9 Hz), 39.54, 39.55, 33.4, 33.2, 30.8 (d, J = 4.0 Hz), 30.7 (d, J = 4.0 Hz), 22.8, 22.7, 22.3, 18.32, 18.29 , 16.5 (d, J = 6.0 Hz). NMR ³¹ P{ ¹ H} (162 MHz, CDCl ₃ , δ ppm): 20.4. IR ν (cm- ¹ ): 2926, 1772, 1435, 1379, 1319, 1257, 1211, 1167, 1120, 1012, 960, 853, 810, 753, 664, 643, 575, 511. Mass spectrum (HRMS- ESI): m/z calculated for C ₃₆ H ₄₉ NO ₉ P ⁺ [M+H] ⁺ : 670.3140, found: 670.3145.

Example 4. Synthesis of dibenzyl(((1a S ,3a S ,4 S ,6a S ,6b R ,9a R )-1a,7-dimethyl-5-oxo-2,3,3a,4,5,6a,6b ,9-octahydro-1a H -oxyreno[2',3':8,8a]azuleno[4,5- b ]furan-4-yl)methyl)phosphonate Ig.

N -Bu ₃ P (0.081 g, 0.4 mmol) was quickly added to a mixture of dibenzylphosphite 6 (1.048 g, 4 mmol) with arglabin 1 (0.984 g, 5 mmol) in acetonitrile (5 ml), and the reaction mixture was kept at 20°C within 1 hour. The solvent is then removed under reduced pressure, and the residue is washed several times with diethyl ether and dried under reduced pressure. Yield: 98% (1.99 g), white powder, _mp. 106-107 °C, [α] _D ²⁷ = +67.4 ( c 2.00; CHCl ₃ ).

NMR ¹ H (400 MHz, CDCl ₃ , δ ppm): 7.39 - 7.31 (m, 10H), 5.54 (br. s, 1H), 5.08 - 4.90 (m, 4H), 3.95 (t, J = 10.1 Hz, 1H), 2.79 - 2.64 (m, overlapping signals 2H), 2.45 (dddd, J = 23.7, 12.4, 5.4, 5.4 Hz, 1H), 2.35 (dddd, J = 19.0, 16.0, 5.4 Hz, 1H) , 2.14 - 2.02 (m, overlapping signals 3H), 1.97 - 1.90 (m, 1H), 1.89 (b.s., 3H), 1.82 - 1.74 (m, 1H), 1.70 - 1.56 (m, 1H), 1.37 - 1.27 (m, 1H), 1.27 (s, 3H). NMR ¹³ C{ ¹ H} (100.6 MHz, CDCl ₃ , δ ppm): 176.9 (d, J = 9.5 Hz), 140.5, 136.0, 128.6 (d, J = 6.9 Hz), 128.3 (d, J = 4.3 Hz), 124.8, 82.8, 72.3, 67.7 (d, J = 6.3 Hz), 67.4 (d, J = 6.3 Hz), 62.6, 52.3 (d, J = 4.5 Hz), 52.2, 41.2 (d, J = 4.4 Hz), 39.5, 33.1, 24.4 (d, J = 144.4 Hz), 22.6, 22.5, 18.3. NMR ³¹ P{ ¹ H} (162 MHz, CDCl ₃ , δ ppm): 29.2. IR ν (cm- ¹ ): 3032, 2932, 1772, 1497, 1451, 1432, 1407, 1384, 1318, 1246, 1231, 1190, 1177, 1119, 1081, 1047, 1025, 991, 970, 919, 866, 856, 807, 784, 735, 717, 696, 674, 663, 646, 614, 597, 576, 538, 520, 494, 464. Mass spectrum (HRMS-ESI): m/z calculated for C ₂₉ H ₃₄ O ₆ P ⁺ [M+H] ⁺ : 509.2088, found: 509.2093.

Example 5. Synthesis of 3-(((1a S ,3a S ,4 R ,6a S ,6b R ,9a R )-1a,7-dimethyl-5-oxo-2,3,3a,4,5,6a, 6b,9-octahydro-1a H -oxyreno[2',3':8,8a]azuleno[4,5- b ]furan-4-yl)methyl)oxazolidin-2-one Id.

n -Bu ₃ P (0.081 g, 0.4 mmol) was quickly added to a mixture of 2-oxazolidinone 7 (0.348 g, 4 mmol) with arglabin 1 (0.984 g, 4 mmol) in acetonitrile (5 ml), and the reaction mixture was kept at 20 °C for 1 hour. The solvent is then removed under reduced pressure, and the product is purified by column chromatography (SiO ₂ , eluent n -hexane/EtOAc=1:1, R _f = 0.71). Yield: 87% (1.16 g), white powder, _mp. 87-88°C, [α] _D ²⁵ = +71.2 ( c 1.00; CHCl ₃ ). NMR ¹ H (400 MHz, CDCl ₃ , δ ppm): 5.58 (br. s, 1H), 4.39 - 4.25 (m, 2H), 4.08 (t, J = 10.2 Hz, 1H), 3.79 - 3.59 (m, 3H), 3.49 (dd, J = 14.8, 3.3 Hz, 1H), 2.87 (d, J = 10.7 Hz, 1H), 2.81 - 2.71 (m, 1H), 2.39 (dt, J = 12.9, 3.7 Hz, 1H), 2.19 - 2.09 (m, 2H), 2.05 - 1.96 (m, 1H), 1.96 - 1.87 (m, 4H), 1.69 - 1.55 (m, 1H), 1.51 - 1.37 (m, 1H), 1.33 (s, 3H). NMR ¹³ C{ ¹ H} (100.6 MHz, CDCl ₃ , δ ppm): 177.1, 159.6, 140.3, 125.1, 83.3, 72.5, 62.9, 62.3, 52.5, 49.1, 46.9, 46.5, 41.3, 39.6 , 33.5 , 22.7, 22.5, 18.3. IR ν (cm- ¹ ): 2929, 1780, 1746, 1735, 1501, 1488, 1437, 1415, 1367, 1326, 1309, 1265, 1231, 1209, 1190, 1168, 1143, 1113, 108 6, 1061, 1029, 999, 964, 946, 930, 897, 871, 822, 795, 769, 745, 689, 665, 650, 618, 596, 568, 510, 498, 459. Mass spectrum (HRMS-ESI): m/z calculated for C ₁₈ H ₂₄ NO ₅ ⁺ [M+H] ⁺ : 334.1649, found: 334.1654.

Example 6. Synthesis of 1-(((1a S ,3a S ,4 R ,6a S ,6b R ,9a R )-1a,7-dimethyl-5-oxo-2,3,3a,4,5,6a, 6b,9-octahydro-1a H -oxyreno[2',3':8,8a]azuleno[4,5- b ]furan-4-yl)methyl)pyrimidin-2,4( 1H , 3H )- Dione Ie.

To a mixture of uracil8 (0.448 g, 4 mmol) with arglabin1 (0.984 g, 4 mmol) in N,N-dimethylformamide (5 ml) quickly addn-Bu₃P (0.081 g, 0.4 mmol), and the reaction mixture was kept at 20°C for 1 hour. The solvent is then removed under reduced pressure and the product is purified by column chromatography (SiO₂, eluent - C₆H₆/EtOAc=1:3,R _f = 0.63). Yield: 97% (1.39 g), colorless crystals, T_pl. 201-202°C (with decomposition), [α]_D ²⁵= +101.5 (c 1.00; CHCl₃). NMR¹H (400 MHz, CDCl₃, δ ppm): 8.71 (s, 1H), 7.52 (d,J = 8.0 Hz, 1H), 5.70 (dd,J = 8.0, 2.3 Hz, 1H), 5.61 - 5.55 (m, 1H), 4.18 - 4.05 (m, 2H), 3.98 (dd,J = 14.5, 3.0 Hz, 1H), 2.89 - 2.70 (m, 2H), 2.56 (ddd,J = 12.7, 5.3, 2.9 Hz, 1H), 2.21 - 2.09 (m, 2H), 2.02 - 1.87 (m, 5H), 1.56 - 1.41 (m, 2H), 1.34 (s, 3H). NMR¹³C{¹H} (100.6 MHz, CDCl₃, δ ppm): 176.4, 163.2, 151.2, 146.0, 140.1, 125.2, 102.4, 83.5, 72.4, 62.8, 52.4, 49.5, 46.7, 44.8, 39.6, 33.5, 22.7, 22. 6, 18.2. IR ν (cm-¹): 3006, 2960, 2923, 2876, 2819, 2051, 1769, 1693, 1661, 1467, 1446, 1431, 1411, 1399, 1377, 1364, 1338, 1319, 1293, 1238, 1 215, 1195, 1184, 1178, 1170, 1150, 1125, 1093, 1062, 1033, 1003, 974, 962, 892, 869, 860, 826, 804, 786, 766, 737, 726, 681, 668, 652, 636, 613, 5 92, 571, 550, 537, 525, 509, 497, 457. Mass spectrum (HRMS-ESI):m/z calculated for C₁₉H₂₃N₂O₅ ⁺ [M+H]⁺: 359.1602, found: 359.1607.

Mild conditions, high speed, high yields of target products, combined with the chemo- and diastereoselectivity of the reactions performed, prove the high efficiency of tri- butylphosphine as a catalyst for the functionalization of natural arglabin using pronucleophilic reagents.

Example 7. Determination of the cytotoxicity of the claimed arglabin derivatives.

Arglabin 1 , its known derivative 2 and new derivatives Ia - Ie have been tested for cytotoxicity against a wide range of cancer and normal cell lines of various origins.

The following cancer cell lines were used:

- M-HeLa clone 11 (epithelioid carcinoma of the cervix, HeLa subline, clone M-HeLa);

- T98G - human glioblastoma;

- HepG2 - human liver carcinoma;

- PANC-1 - human pancreatic carcinoma;

- HuTu 80 - adenocarcinoma of the human duodenum;

- MCF-7 - human breast adenocarcinoma (pleural fluid);

- A 549 - human lung carcinoma;

- PC3 - prostate adenocarcinoma cell line from ATCC (American Type Cell Collection, USA; CRL 1435);

- SK-OV-3 - human ovarian adenocarcinoma;

- DU-145 - human prostate carcinoma;

- A 375 - human skin melanoma from the CLS Cell Lines Service cell repository.

From normal cell lines we used:

- WI38 - VA 13 subline 2RA - human embryonic lung from the collection of the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg);

- Chang liver - human liver cells from the collection and Research Institute of Virology of the Russian Academy of Medical Sciences (Moscow).

The cytotoxic effect on cells was determined using the colorimetric method of cell proliferation - the MTT test. NADPH-dependent cellular oxidoreductase enzymes can, under certain conditions, reflect the number of viable cells. These enzymes are capable of reducing the tetrazolium dye (MTT) - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide into an insoluble blue-violet formazan, which crystallizes inside the cell. The amount of formazan formed is proportional to the number of cells with active metabolism.

Cells were seeded onto a 96-well plate from Nunc at a concentration of 5×10 ³ cells per well in a volume of 100 μl of medium and cultured in a CO ₂ incubator at 37°C until a monolayer was formed. Then the nutrient medium was removed and 100 μl of solutions of the test compound in specified dilutions were added to the wells, which were prepared directly in the nutrient medium with the addition of 5% dimethyl sulfoxide to improve solubility. Studies were carried out in a concentration range (1-100 µM). After 48 h of incubation of cells with test compounds, the culture medium was removed from the plates and 100 μl of serum-free culture medium with MTT at a concentration of 0.5 mg/ml was added and incubated for 4 h at 37°C. At the end of incubation, the medium with MTT was removed and 100 μl of dimethyl sulfoxide was added to each well to dissolve the formed formazan crystals. Optical density was recorded at 540 nm on an Invitrologic microplate reader (Novosibirsk, Russia). Experiments for all compounds were repeated three times.

Calculation of IC ₅₀ - the concentration of the test compound that causes inhibition of cell growth by 50%, was carried out using the program: MLA - “Quest Graph™ IC50 Calculator” [MLA - “Quest Graph™ IC50 Calculator”. AAT Bioquest, Inc, January 26, 2022, https://www.aatbio.com/tools/ic50-calculator]. The new class of compounds is considered cytotoxically active with an IC ₅₀ <100 μM [Guide to preclinical drug studies. Part 1. / ed. Mironova A.N. - M.: 2012, 944 pp.].

Table 1 in Figure 1 presents the IC ₅₀ values in μM of arglabin and its derivatives. From the data obtained it is clear that arglabin 1 and its derivative 2 exhibit a cytotoxic effect against all tumor lines used in the experiments in the IC ₅₀ concentration range from 11.3 to 28.0 µM. However, in relation to normal cell lines Chang liver and WI38, the cytotoxicity of compounds 1 and 2 is also very high (12.0 and 8.5 μM for 1 and 10.0 and 32.0 μM for 2 , respectively), that is, they do not have a selective effect against cancer cells, which corresponds to the existing level of development of medicinal chemistry.

The claimed derivatives of arglabin Ia - Ie also showed a cytotoxic effect over the entire range of concentrations studied. Compounds Ia , Ib , Ig and Id showed the highest cytotoxicity against duodenal adenocarcinoma HuTu 80 (13.3, 26.9, 16.4 and 14.5 µM, respectively); compound Ib - against breast adenocarcinoma MCF-7 (18.3 µM), glioblastoma T98G (16.9 µM) and melanoma A 375 (19.8 µM); compounds Ig and Ie - against cervical carcinoma M-HeLa (10.3 and 21.6 µM, respectively). It should be separately emphasized the high (compared to compounds 1 and 2 ) cytotoxic effect of derivative Ib (16.9 μM) against the T98G glioblastoma line, which is currently the most aggressive fast-growing malignant human brain tumor.

As noted above, an important criterion for assessing the cytotoxic effect is also the selectivity of the compound against cancer cells. To assess the cytotoxic effect, selectivity indices (SI) were calculated as the ratio of the IC ₅₀ value for normal liver cells (Chang liver) and the IC ₅₀ value for cancer cells (Table 2). Compounds with SI≥3 are considered selective [11].

As can be seen from Table 2, compounds Ia and Id are highly selective against duodenal adenocarcinoma HuTu 80 with selectivity indices of 4.7 and 5.4, respectively; compound Ib has lower selectivity against this type of tumor and is 1.5. Compound Ie is selective against the M-HeLa cancer line (SI=3.3). Compound Ig is selective towards two cancer lines - M-HeLa (SI=5.0) and HuTu 80 (SI=3.2). Compound Ib exhibits selectivity against three tumor lines at once: MCF-7 (SI = 3.0), T98G (SI = 3.2) and A 375 (SI = 2.7). At the same time, reference compounds 1 and 2 are significantly inferior to the leading compounds in selectivity (SI≤1).

Thus, the practical results obtained confirm the validity of the accepted working hypothesis and prove the presence of a selective cytotoxic effect in arglabin derivatives Ia - Ie , which can subsequently be used to create targeted antitumor drugs.

Thus, from the above we can conclude that the applicant has achieved the stated technical result, namely: a product has been developed based on the arglabin derivative of formula Ia , which has a selective cytotoxic effect against duodenal adenocarcinoma HuTu 80.

The claimed technical solution complies with the patentability condition of “novelty” imposed on inventions, since no technical solutions have been identified from the researched level of technology that have the declared set of distinctive features that ensure the achievement of the declared results.

The claimed technical solution complies with the patentability condition “inventive step” imposed on inventions, since the applicant for the first time identified a compound that has high cytotoxicity towards cells of predominantly malignant neoplasms, which is not obvious to a specialist in this field of science and technology.

The claimed technical solution meets the “industrial applicability” criterion for inventions, since it can be implemented at any specialized enterprise using standard equipment.

Claims (2)

Use of an arglabin derivative of formula Ia as an agent with a selective cytotoxic effect against duodenal adenocarcinoma HuTu 80 .