LV12403B - Cytotoxicaly derivatives of penicillanic acid - Google Patents

Abstract

The present invention pertains to the pharmacologically active substances, particularly to cytotoxically agents for chemotherapy for cancer. The said compounds contain modifying penicillin sulfoxide esters or penicillin sulfone esters having specifically selected substituents in the position 6 and in the 2-beta-methylgroup. According results of experiments the invented substances have cytotoxically quality in vitro on cell-lines HT 1080, MG-22A and B 16: by using law-concentration of said substances there is observable death of 50 % cells. There is observed direct correlation between cytotoxically activity of studied compounds and their ability to induce biosynthesis of NO in cells. Compounds having expressed cytotoxically activities have considerably extent the mentioned ability to induce biosynthesis.

Description

CYTOTOXIC PENICYLANIC ACID DERIVATIVES

The present invention relates to novel pharmacologically active substances, in particular to cytotoxic preparations for cancer chemotherapy containing penicillanic acid sulfoxides or sulfones with specially selected substituents in the penam cycle at positions 2 and 6. in synthesis [1-3]. However, no substances with cytotoxic activity have been found in this class of chemical compounds.

The object of the invention is the synthesis of novel compounds which are characterized by high cytotoxic activity due to enhanced nitric oxide biosynthesis in malignant cells. It is a novel anticancer mechanism that distinguishes penicillin sulfoxides and sulfones from known anticancer agents which have an alkylating, antimetabolic, immunostimulatory or hormonal action [4],

This objective was achieved by using already known cytotoxic agents, as well as the novel poly-substituted penicillin sulfoxide and sifone esters obtained according to Scheme 1, which include the following reactions:

• cyclization of the unsubstituted and substituted 2- [4- (heterildithio) -2-oxo-azetidinyl-1] -2- (zzopropenyl) -acetates at the 3-position to 2P-monomethyl-substituted penicillates;

• condensation of 6-oxo-penicillanic acid esters 5 with phosphorylides 6;

• oxidation of 2P-monomethyl substituted penicillanates and 6-alkylidene penicillanates with KMnO ₄ or

3-Chlorobenzoic acid (3-HBPS) to sulfoxide or sulfone 2-4, 7.

The cytotoxic activity of the patented compounds was tested in vitro on four cell lines: MG-22A (murine hepatoma), HT-1080 (human fibrosarcoma), Neiro 2A (murine neuroblastoma) and B16 (murine melanoma). The results obtained are shown in Tables 1 and 2 The cytotoxic effect was evaluated by calculating the number of cells killed by the test compound in the immunological plates using two colorimetric methods:

• staining with crystal violet CV (dye is absorbed by living cell membranes) • staining with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide MTT (dye absorbed by living cell mitochondrial enzymes).

The results of the experiment show that the cytotoxic effect (TD 50) of compounds 2-4 on HT 1080, MG-22A cell lines is observed at concentrations of 12-100 pg / ml (see Table 1). For 6-alkylidene penicins 7a-d this effect is most pronounced: 50% cell death of HT 1080, MG-22A and B 16 is observed at cytotoxic concentrations (TD ₅₀ ) of 1-12 pg / ml (see Table 2). For the Neiro 2A cell line, cytotoxic effects were observed only for compound 7d.

Nitric oxide molecules, which are formed in cells of the body, are known to have pronounced cytotoxic effects [5]. The ability to intensify NO biosynthesis is characteristic of all patentable penicillin derivatives (see Tables 1 and 2) and is characterized by TGioo, which is calculated by the following formula:

TGioo = G _ex · 100 / C (ηΜ · 10'7200μ1) where: TGioo is the specific capacity of the compound to generate NO biosynthesis, which is extrapolated to 100% of living cells;

Gex - NO concentration (nM) in biological medium, determined at 200 μΐ (plate-well volume) after incubation with test substance (10 or 100 pg / ml);

C = percentage of surviving cells after incubation with test substance (using CV staining).

Scheme 1

a-c

a) R = H, R ¹ = CHPh ₂

b) R = Cl, R ¹ = CHPh ₂

c) R = Cl, R ¹ = / - Bu

1. ClCH ₂ COOH, CH ₃ COOAg

a-c

a) R = H, Hal = Cl

b) R = Cl, Hal = Cl

c) R = Cl, Hal = Br

COOR!

a, b

a) R ¹ = CHPh ₂

b) R ¹ = _i C ₄ H _g

1. R ² CH = PPh ₃

2. 3-HBPS

COOR a-d

a) R ¹ = CHPh ₂ , R ² = / - BuOCO, n = 1

b) R ¹ = CHPh ₂ , R ² = / - BuOCO, n = 2

c) R ¹ = / - Bu, IT = / - BuOCO, n = 2 ·

d) R ¹ = / - Bu, R ² = Ph, n = 2

Table 1. Cytotoxic effects of Penicillan Scab Esters and their Effect on Cancer Cell Ability to Generate NO in vitro

Savie canopy Cell lines HT-1080 MG-22A td ₅₀ (CV) ³ TDso (MTT) ^b TG _1oo ^C td ₅₀ (CV) td ₅₀ (MTT) TGioo 2a 50 50 850 50 50 243 2b 100 > 100 61 > 100 > 100 41 3a 100 fifty three 800 60 90 142 3b 13th 46 1100 40 42 467 3c 9th 42 800 45 34 797 4 29th 12th 900 45 28th 950

Concentration of ³ substances (pg / ml) causing 50% cell death as determined by CV staining ^b Concentration (pg / ml) causing 50% cell death as determined by MTT staining ^c specificity generate NO

Experimental part

Penicillins 2-4 and 7 were obtained by the following methods;

Example 1

Benzhydryl-2P-benzothiazolylsulfonylmethyl-2a-methylpenamine-3a-carboxylate sulfone (2a) Benzhydryl-2- [4- (2-benzothiazolylthiothio) -2-oxo-azetidinyl-1] -2- (zzo-propenyl) -acetate (340) mg, 0.51 mM) was added BF ₃ -Et ₂ O (0.05 mL) in 14 mL of dichloromethane in nitromethane (1: 1). The reaction mixture is allowed to stand for 24 hours at room temperature and the solvents are evaporated under reduced pressure. The residue (0.17 g) is dissolved in acetic acid (3.5 mL), water (1.0 mL) and KMnO ₄ (600 mg, 3.8 mM) are added. Suspension stirred for 2 hours at room temperature, cool in an ice water bath and _KMnO4 superiority reduced using hydrogen peroxide diluted with water (30 ml) and extracted with dichloromethane (30 ml). The organic phase is washed with saturated NaHCO ₃ solution, dried over Na ₂ SO ₄ . Dichloromethane is distilled off under reduced pressure, and the residue is chromatographed on a Merck Kieselgel (0.063-0.230 mm) column (eluent: benzene-acetone, 4: 1). 100 mg (54%) of benzhydryl-23-benzothiazolylsulfonylmethyl-2a-methylpenamine-3a-carboxylate sulfone are obtained, m.p. 170-171 ° C. Assay Content: 93% (by HPLC).

PMR (CDCl ₃ ), δ: 1.52 (3H, s, CH ₃ ); 3.48 (2H, m, 6-H); 3.90, 4.15 (2H, AB-q, > 14, CH ₂ SO ₂ ); 4.59 (1H, d, d,>2,> 4, 5-H); 5.28 (1H, s, 3-H); 6.85 (1H, CHPh ₂ ); 7.15 to 7.44 (10H, m, 2C6H _5); 7.48-8.28 (4 H, m, Ce H).

Discover %: C, 56.55; H, 4.14; N, 4.64. C ₂₈ H ₂₄ N ₂ O ₇ S ₃ . Apr. %: C, 56.36; H, 4.05; N, 4.68.

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Example 2

Benzhydryl-23-benzothiazolylsulfonylmethyl-6a-chloro-2a-methylpenamine-3a-carboxylate sulfone (2b)

The compound is synthesized from benzhydryl-2- [4- (2-benzothiazolyldithio) -3-chloro-2-oxo-azetidinyl-1] -2- (2-isopropyl) -acetate (500 mg, 0.96 mM) according to Example 1. Example 1 using ethyl acetate-hexane (1: 2) as the eluent for column chromatography. 40 mg (6.6%) of benzhydryl-2-benzothiazolylsulfonylmethyl-6a-chloro-2a-methylpenam-3a-carboxylate sulfone are obtained, m.p. 57-59 ° C. Content of the basic substance 97% (based on AESH).

PMR (CDCl ₃ ), δ: 1.37 (3H, s, CH ₃ ); 3.67, 4.13 (2H, AB-q, J = 14, CH ₂ S); 5.04 (1H, s, 3-H); 5.13 (1H, d J = 1Hz, 5-H); 5.40 (1H, d J = 1Hz, 6-H); 7.00 (1H, CHPh ₂ ); 7.22-8.00 (14H, m, 2CeH5, C6H4).

Discover %: C, 53.43; H, 4.03; N, 4.15. C ₂₈ H23C1N ₂ O ₇ S ₃ Apr. %: C, 53.28; H, 3.67; N, 4.43.

Benzhydryl-2P-chloromethyl-2a-methylpenam-3a-carboxylate sulfone (3a) was synthesized according to method [2],

Benzhydryl-2P-chloromethyl-6a-chloro-2a-methylpenam-3a-carboxylate sulfone (3b) was synthesized according to method [2].

Benzhydryl-2β-bromomethyl-6α-chloro-2α-methylpenamine-3α-carboxylate sulfone (3c) was synthesized according to method [2],

Example 3

Benzhydryl-2P-chloroacetoxymethyl-6-chloro-2a-methylpenamine-3a-carboxylate sulfone (4)

To a suspension of silver acetate (0.84 g, 5 mM) in dichloromethane (40 mL) was added chloroacetic acid (8.40 g, 87.5 mM) and benzhydryl-2- [4- (2-benzothiazolyldithio) -3-chloro-2- oxo-azetidinyl-1] -2- (2-isopropenyl) acetate (1.46 g, 2.5 mM) in dichloromethane (5 mL). The reaction mixture is allowed to stand for 4 hours at room temperature. The suspension is filtered through a silica gel column. The organic phase is washed with a saturated solution of NaHCO ₃ , dried over Na ₂ SO 4 and distilled under reduced pressure. The residue was poured into ether (2 mL) and evaporated to give 820 mg (64%) of crystalline benzhydryl-2P-chloroacetoxymethyl-2a-methylpenam-3a-carboxylate. Content of the basic substance 80% (by HPLC).

To a solution of benzhydryl-2P-chloroacetoxymethyl-2a-methylpenam-3a-carboxylate (500 mg, 1.15 mM) in acetic acid (10 mL) was added water (1.7 mL) and KMnO4 (320 mg, 2.02 mM). The suspension is stirred at room temperature for 3 hours, cooled in an ice-water bath and the excess KMnO ₄ is reduced with hydrogen peroxide. The reaction mixture was diluted with water and dichloromethane. The organic phase is washed with saturated solutions of NaHCO ₃ and NaCl, dried over Na ₂ SO 4. Evaporate the solution under reduced pressure. Chromatograph the residue on a column of Merck Kieselgel (0.063-0.230 mm), eluent: hexane: ethyl acetate (3: 1). Obtain 232 mg (40%) of benzhydryl-2P-chloroacetoxymethyl-2a-methylpenam-3a-carboxylate sulfone, m.p. 57-59 ° C. Content of basic material 98% (by HPLC).

PMR (CDCl ₃ ), δ: 1.33 (3H, s, CH ₃ ); 3.78, 4.00 (2H, d, d, J = 12, CH ₂ Cl); 4.71 (1H, d J = 1 Hz, 5-H); 4.80 (1H, s, 3-H); 5.17 (1H, d J = 1Hz, 6-H); 6.97 (1H, CHPh ₂ ); 7.40 (10H, m, 2C ₆ H ₅ ).

Example 4.

Benzhydryl-6 (Z) -tert-butoxycarbonylmethylene penicillanate 1-oxide (7a).

A solution of benzhydryl-6 (Z) - / erobutoxycarbonylmethylene penicillanate [3] (500 mg, 1.04 mM) in dichloromethane (20 mL) was cooled to O'C, added with 3-chlorobenzoic acid (180 mg, 1.04 mM) and stirred for 1 hour. at 0 C and 1 hour at room temperature. The reaction mixture is washed with saturated solutions of NaHCO ₃ and NaCl, dried over Na ₂ SO 4. Evaporate the solution under reduced pressure. Chromatograph the residue on a Merck Kieselgel (0.063-0.230 mm) column, eluting with ethyl acetate-hexane (1: 1). 250 mg (48%) of benzhydryl-6 (Z) - tert -butoxycarbonylmethylene penicillanate sulfoxide are obtained, m.p. 179-181 ° C.

PMR (CDCl 3), δ: 0.98 (3H, s, 2-CH ₃ ); 1.51 (9H, s, C 4 H 9); 1.67 (3H, s, 2-CH _3); 4.78 (1H, s, 3-H); 5.68 (1H, d, J = 2.5-H); 6.49 (1H, d,> 2, -CH =); 7.01 (1H, CHPh ₂ ); 7.34 (10H, s, 2 C ₆ H ₅ ).

Discover %: C, 56.55; H, 4.14; N, 4.64. C ₂₈ H ₂ 4N ₂ O ₇ S ₃ Apr. %: C, 56.36; H, 4.05; N, 4.68.

Benzhydryl-6 (Z) -carbutoxycarbonylmethylene-penicillanate sulfone (7b) was synthesized according to method [3],

Example 5.

7ert-Butyl-6 (Z) -tert-butoxycarbonylmethylene-penicillanate sulfone (7c).

A solution of 7-tert-butyl-6-oxo-penicillanate (1.00 g, 3.69 mM) and terobutoxycarbonylmethylene triphenylphosphorane (1.60 g, 4.43 mM) in dichloromethane (30 mL) was stirred at room temperature for 2 hours. The solvent is evaporated off under reduced pressure. Chromatograph the residue on a Merck Kieselgel (0.063-0.230 mm) column, eluting with ethyl acetate-hexane (1: 1). 250 mg (15%) of a mixture of Z and E isomers of tert-butyl 6-tert-butoxycarbonylmethylene penicillate are obtained.

A solution of 7-tert-butyl-6-tert-butoxycarbonylmethylene penicillanate (200 mg, 0.54 mM) in dichloromethane (20 mL) was cooled to 0 DEG C., added with 3-chlorobenzoic acid (400 mg, 2.32 mM) and stirred for 1 hour at 0 '. C and 1 hour at room temperature. The reaction mixture is washed with saturated solutions of NaHCO ₃ and NaCl, dried with Na ₂ SC> 4. Evaporate the solution under reduced pressure. Chromatograph the residue on a Merck Kieselgel (0.063-0.230 mm) column, eluting with ethyl acetate: hexane (1: 1). 120 mg (57%) of tert-butyl-6 (Z) -tert-butoxycarbonylmethylene penicillanate sulfone are obtained, m.p. 138-140 ° C. Content 90% (by HPLC).

PMR (CDCl ₃ ), δ: 1.28 (3H, s, CH ₃ ); 1.44-1.66 (21H, m, CH ₃ , 2C ₄ H 9); 4.42 (1H, s, 3-H); 5.46 (1H, d, J = 2.5, H); 6.51 (1H, d, J = 2, -CH =).

Example 6.

Tert-butyl 6-benzylidene penicillanate sulfone (7d).

Sodium hydride (84 mg, 3.5 mM) was added to a solution of 7c-butyl-6-oxopenicyanate (0.80 g, 2.95 mM) and benzylidene triphenylphosphonium chloride (2.60 g, 5.95 mM) in dichloromethane (40 mL). The solution is stirred at room temperature for 2 hours, washed with dilute HCl, dried with Na ₂ SO 4. The solvent is evaporated off under reduced pressure. Chromatograph the residue on a column of Merck Kieselgel (0.063-0.230 mm), eluting with ethyl acetate / petroleum ether (1: 2). 500 mg (49%) of a mixture of Z and E isomers of Zert-butyl-6-benzylidene penicillate (3: 1) are obtained.

A solution of 7-tert-butyl-6-benzylidene penicillanate (300 mg, 0.87 mM) in dichloromethane (20 mL) was cooled to 0'C, added with 3-chlorobenzoic acid (450 mg, 2.61 mM) and stirred for 1 hour at 0'C and 1 hour at room temperature. The reaction mixture is washed with saturated solutions of NaHCO ₃ and NaCl, dried over Na ₂ SO 4. Evaporate the solution under reduced pressure. Chromatograph the residue on a Merck Kieselgel (0.063-0.230 mm) column, eluting with ethyl acetate-hexane (1: 2). 100 mg (32%) of a mixture of Z- and E-isomers of the tert-butyl 6-benzylidene penicillanate sulfone are obtained (5: 1). Content of basic material 98% (by HPLC).

Z-isomer. PMR (CDCl ₃ ), δ: 1.40-1.67 (15H, m, 2CH ₃ , C 4 H 9); 4.38 (1H, s, 3-H); 5.43 (1H, d, J = 0.5, 5-H), 7.40 (1H, d, J = 0.5, -CH =); 7.40-7.58 (3H, m, C ₆ H ₅ ); 7.92-8.00 (2H, m, C ₆ H ₅ ).

E-isomer. PMR (CDCl ₃ ), δ: 1.40-1.67 (15H, m, 2CH ₃ , C ₄ H ₉ ); 4.59 (1H, s, 3-H); 5.14 (1H, br.s, 5-H); 6.84 (1H, br.s, -CH =); 7.40-7.58 (5H, m, C ₆ H ₅ ).

Determination of cytotoxic effects in vitro

Solutions of test compounds in dimethyl sulfoxide were injected into 96-well plates together with cells. Cells (3x10 ⁴ cells / ml) were cultured for 72 hours in DMEM medium without indicator and antibiotics. After thawing the ampoules, the cells were transfused no more than four times. Control cells without culture were cultured on a separate plate. The cytotoxic effect was evaluated by the number of surviving cells as determined by two colorimetric methods:

a) staining with crystal violet CV (the dye is absorbed on the membranes of living cells),

(b) staining with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) MTT (the dye is absorbed by the mitochondrial enzymes of living cells) [6],

The amount of live cells in the control panel was assumed to be 100%.

The concentration of nitric oxide in the presence of test substances was determined by the Grace method [7],

We patent:

1. Penicillanic acid derivatives of the general formula:

wherein: R and R ', taken together or individually, are hydrogen, halogen or' substituted methylene, Y is hydrogen, halogen, acyloxy or 2-benzothiazolylsulfon, R ² is diarylalkyl or alkyl, n = 1, 2.

Structural analogs of penicillin sulfone according to pl, wherein R and R ^1, taken together or separately, are substituents selected from the group consisting of hydrogen and chlorine; Y is 2-benzothiazolylsulfone and R ² is benzhydryl.

3. Penicillin sulfone structural analogues according to pl, wherein R and R ¹ taken together or separately are substituents selected from the group consisting of hydrogen and chlorine; Y is chloroacetoxy; and R ² is benzhydryl.

Structural analogs of penicillin sulfoxide or sulfone according to pl, wherein R and R ¹ together are substituents selected from the group consisting of alkoxycarbonylmethylene or arylmethylene and R ² is diarylalkyl or alkyl.

Structural analogs of penicillin sulfone according to pl, wherein R and R ¹ together are substituents selected from tert-butoxycarbonylmethylene or benzylidene and R ² is tert-butyl.

The structural analogue of penicillin sulfoxide or sulfone according to p.l. use in the treatment of tumors.

Use of penicillin structural analogues after pl for the treatment of tumors wherein: R and R ^{1 =} H, H and Cl, PhCH =; / -B «COOCH =; Y = H, Cl, C1CH2COO, SO2-Het; Het = 2-benzothiazolyl; R ² = CHPh ² , r-Bu; n = 1, 2.

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Literature

1. Danelon G.O., MataE.G., Masmaretti O.A. Bioorg. Med. Chem. Lett., 1995, vol.5,

No. 17, 2037-2040.

Taiho Pharmaceutical Co., Ltd. JP 58.04,788; CA, 1983, vol.98, 197889z.

Buynak, J.D .; Geng, B ,; Bachman, B.; Hua, L. Bioorg. Med. Chem. Lett., 1995, vol.5, no. 14, 1513.

4. Editors: Storelli, A.C .; Johns, D.C. Antineoplastic and Immunosuppressive Agent Part II. Springer-Verlag Heidelberg, New York, 1975.

Kervvin J. F Jr., Lancaster F.R.Jr .; Feldman P.L. JMed Chem, 1995, vol.38, 4343-4362.

Fast D.J .; Lynch, R.C .; Leu R.W. JLeuckocyt. Biol., 1992, vol.52, 255-261.

Freshliey P.J.; Culture of Animal Celis (A Manual of Basic Technique), Wiley-Liss, NewYork, 1994, 296-297.

Claims (7)

1. Penicillanic acid derivatives of the general formula: wherein: R and R ^1, taken together or individually, are hydrogen, halogen or substituted methylene, Y is hydrogen, halogen, acyloxy or 2-benzothiazolylsulfon, R ² is diarylalkyl or alkyl, n = 1, 2. Structural analogs of penicillin sulfone according to pl, wherein R and R ^1, taken together or separately, are substituents selected from the group consisting of hydrogen and chlorine; Y is 2-benzothiazolylsulfone and R ² is benzhydryl. 3. Penicillin sulfone structural analogues according to pl, wherein R and R ¹ taken together or separately are substituents selected from the group consisting of hydrogen and chlorine; Y and chloroacetoxygrapa; and R ² is benzhydryl. Structural analogs of penicillin sulfoxide or sulfone according to pl, wherein R and R ¹ together are substituents selected from the group consisting of alkoxycarbonylmethylene or arylmethylene and R ² is diarylalkyl or alkyl. Structural analogs of penicillin sulfone according to p 1, characterized in that R and R ¹ together are a substituent selected from tert-butoxycarbonylmethylene or benzylidene and R ² is a tert-butyl group. The structural analogue of penicillin sulfoxide or sulfone according to p.l. use in the treatment of tumors. Use of penicillin structural analogues after pl for the treatment of tumors, wherein: R and R ^{1 =} H, H and Cl, PhCH =; Z-5 «C00CH =; Y = H, Cl, C1CH2COO, SO2-Het; Het = 2-benzothiazolyl; R ² = CHPh ² , f-Bu; n = 1, 2.