Synthesis and Сytotoxicity of A-Azepanobetulinic Acid N -Methyl-Piperazinylamide

Abstract

The synthesis of A-azepanobetulinic acid N-methylpiperazinylamide was performed through a series of transformations (oximation, Beckmann rearrangement, reduction) of previously synthesized betulonic acid N-methylpiperazinylamide. In vitro cytotoxic activity was detected for the obtained compound against a number of cancer cell lines, and its potential was revealed as an antibacterial agent.

Pentacyclic triterpenoids are a class of natural compounds widely represented in plant sources. The best-known representatives are betulinic, oleanolic, ursolic, and glycyrrhetinic acids. As evident from literature data, triterpenoids are characteristic of a diverse biological activity, especially antitumor and antimicrobial. For example, betulinic acid is in stage II clinical trials as an antitumor agent against melanoma. ¹ Oleanolic acid shows high activity against hepatocellular carcinoma. ² Besides, oleanolic and ursolic acids are well-known agents against Mycobacterium tuberculosis. ³

Most terpene structures are characterized by common drawbacks, including low solubility, poor bioavailability, and instability to metabolism. ⁴ In addition, they have a weak pharmacological effect at low concentrations. ⁵ As a result, investigations into the chemical modification of the initial carbocyclic backbone have been undertaken in order to find biologically active compounds with an improved pharmacological profile and advanced mechanism of action against tumor cells. Various methods of triterpenoid molecule delivery to biological targets are being actively studied. ⁶

One of the important pharmacophore groups in the triterpenoid modifications is the piperazinyl fragment responsible for various biological activities: antibacterial, ⁷ anticancer, ^8,9 antiviral, ^10,11 antimalarial, ¹² α-glucosidase inhibitory, ¹³ antileishmanial, ¹⁴ antifungal, ¹⁵ etc.

This paper reports on the synthesis and study of the cytotoxicity and antibacterial activity of a new triterpene conjugate with N-methylpiperazine. Based on previously obtained data, concerning the antitumor and antituberculosis activity of azepanotriterpene alcohols and their derivatives, ^16,17 we carried out the transformation of the carbocyclic ring A of betulonic acid N-methylpiperazinylamide 1 to a 7-member A-aza-ring (Scheme 1). At the first stage, compound 1 was transformed to oxime 2, and then converted to lactame 3 via the Beckmann rearrangement. The opening of lactame ring and formation of A-seco-nitrile as a byproduct (the structure is not shown) was also observed in minor amount under TLC monitoring and separated through crystallization of the target lactame. The reduction of compound 3 by LiAlH₄ in THF resulted in A-azepanobetulinic acid N-methylpiperazinylamide 4. The structure of compounds 2 -4 were determined by nuclear magnetic resonance (NMR) spectroscopy. The spectra of all compounds contained signals of amide bond (δ 173.4-174.1 ppm of ¹³C NMR) and N-methylpiperazinyl fragment (δ 2.06-2.45, 2.29, 2.32, 2.51-3.20 ppm of ¹H NMR).

Scheme 1

Synthesis of betulonic acid N-methylpiperazinylamide derivatives 1-5. Reagents and conditions: I. NH₂OH·HCl, EtOH, NaOAc, reflux, 2 hours; II. SOCl₂, dioxane, 0.5 hours, rt; III. LiAlH₄, THF, reflux, 1 hour; IV. NaBH₄, i-PrOH, rt, 2 hours.

In addition, the NMR spectra definitely showed the modification of ring A. For example, the signal of C3 in the spectra of lactame 3 was observed at δ 176.7 ppm, while the conversion to azepane ring led to strong field signal shift to δ 62.3 ppm.

Compounds 1 -5 were tested for antibacterial properties by measuring MIC (minimum inhibitory concentration) of the compound for BW25113 Escherichia coli and BW25113 E. coli ΔtolC (JW5503 from Keio collection). This strain lacks tolC gene and has problems with the efflux system, and it is more sensitive to different antibiotics. ¹⁸ BW25113 E. coli was completely resistant to compound 4, meanwhile ΔtolC strain was sensitive and MIC was 12.5 ± 2.5 µg/mL. MIC for well-known antibiotic erythromycin was 2.5 ± 0.5 µg/mL (Table 1). Compounds 1 -3 and 5 were inactive under the indicated screening conditions. Hence, the presence of the 7-member A-aza-ring is critical for the antibacterial effect against E. coli strains in this case. An interesting fact was the difference in activity between compounds 3 and 4. Moderate values of the antibacterial effect of compound 4 give basis for its further chemical modification.

Table 1

Antibacterial Test Results for Compounds 1-5, MIC µg/mL.

Compds	BW25113 E. coli	Sd	BW25113 E. coli ΔtolC	Sd
1	>100	-	>100	-
2	>100	-	>100	-
3	>100	-	>100	-
4	>100	-	12.5	2.5
5	>100	-	>100	-
Erythromycin	50	2	2.5	0.5

Next, pDualrep2 system was used to analyze the mechanism of action of 4, ¹⁹ but as distinct from erythromycin, or levofloxacin, compound 4 did not induce any reporter gene (data not shown). Thus, it is possible to suggest that it does not inhibit translation or DNA synthesis.

At the second stage, compound 4 was examined in cytotoxicity assay in vitro. The activity determination (CC₅₀ values) was performed using the MTT test. Paclitaxel was used as a reference drug. Compound 4 was tested against LNCaP, PC3, 22RW1, Huh7, and VA13 cell lines and showed a high level of activity in the range 0.2-1 µM (Table 2).

Table 2

Cytotoxicity Test Results for Compounds 1-5, СC₅₀ µM.

Compds	LNCAP	PC3	22RW1	Huh7	VA13
4	1.12	1.11	0.37	0.74	0.19
5	N/a	3.58	N/a	2.75	N/a *
Paclitaxel	0.01	0.015	0.016	0.002	0.001

^* N/a, testing was not performed.

We speculate that key factor in this case is the presence of the N-methylpiperazinylamide fragment in the structure of 4. As a result, the compound obtained is an attractive example for the advanced researches, which is the goal of our ongoing research.

Moreover, we evaluated the activity of compound 5 against cell lines of hepatocarcinoma Huh7 and prostate cancer PC3. Due to the presence of 3-OH group this derivative can be esterified and used for target delivery to cancer cells via prodrug-type conjugates. ^20,21 N-methylpiperazinylamide 5 also demonstrated high values of cytotoxicity, although appeared slightly less active in compare with 4.

Finally, the synthesis of A-azepanobetulinic acid N-methylpiperazinylamide was performed through a series of betulonic acid N-methylpiperazinylamide transformations (oximation, Beckmann rearrangement, reduction). As a result, obtained compound demonstrated its high-value activity against various tumor cell lines, as well as antibacterial potential. High antitumor effect of N-methylpiperazinylamide derivative of betulinic acid suggests the possibility of its use as a precursor for a variety of prodrug-like conjugates.

Experimental

General

NMR spectra were recorded at the Center for Collective Use “Chemistry” of the UIC UFRS RAS. NMR spectra were recorded on a “Bruker AM-300” spectrometer at 300 (¹H) and 75.5 MHz (¹³C) at 25°C. Chemical shifts were given in (δ in ppm) relative to the residual signals of CDCl₃ (¹H 7.24 ppm and ¹³C 76.90 ppm). Melting points were determined with a Boetius heating stage. Optical rotations were measured on Perkin-Elmer 241 polarimeter at room temperature (20°C to 22°C). Combustion analysis was performed with CHNSO-analyser (Model Euro EA 3000, Germany). TLC analysis were conducted on Sorbfil plates (Sorbpolimer, Russian Federation) using chloroform-ethylacetate, 40:1 (v/v). The substances were detected by a 10% solution of H₂SO₄ with subsequent heating at 100°C to 120°C for 2 to 3 minutes. Al₂O₃ (Reakhim, Russian Federation) was used for column chromatography. All the organic solvents were of analytical quality. Compounds 1 and 5 were obtained according to. ⁷

Synthesis of Compound 2

A mixture of compound 1 (0.54 g, 1 mM) and hydroxylamine hydrochloride (0.54 g, 8 mM) in EtOH (15 mL) and NaOAc (0.65 g, 8 mM) was refluxed for 2 hours, poured into H₂O (100 mL), the precipitate was filtered, washed, and dried. The residue was crystallized from EtOH.

3-Oximino-17-(4-Methylpiperazin-1-yl)-Carbonylolup-20(29)-ene (2)

Yield: 0.42 g, 76%, as a white solid.

R _f 0.14.

MP: 192°С.

Anal. Calcd. for С₃₅Н₅₇N₃O₂ (Мr 551.839): С 75.72; Н 10.03; N 7.16. Found: С 76.18, Н 10.41, N 7.61.

¹H NMR (300 MHz, CDCl₃): δ 0.86 (3H, s, H-25), 0.92 (3H, s, H-26), 0.96 (3H, s, H-24), 1.02 (3H, s, H-27), 1.12 (3H, s, H-23), 1.22-2.12 (21Н, m, H-1, H-2, H5-H7, H-9, H11-H13, H-15, H-16, H-18, H-21, H-22), 1.67 (3Н, s, H-30), 2.06-2.18 (4Н, m, H-3′, H-5′), 2.29 (3Н, s, H-7′), 2.32-2.56 (3Н, m, H-13, H-16), 2.84-3.20 (7Н, m, H-19, H-3, H-2′, H-6′, ОН), 4.57 (1H, s, H-29), 4.73 (1H, s, H-29), 9.21 (1H, s, N-OH).

¹³C NMR (CDCl₃, 75.5 MHz): δ 14.5 (C-27), 15.8 (C-25), 15.9 (C-26), 19.3 (C-11), 19.6 (C-30), 20.9 (C-6), 21.6 (C-24), 22.9 (C-23), 25.6 (C-12), 26.5 (C-2), 29.7 (C-15), 31.3 (C-21), 32.4 (C-16), 33.6 (C-22), 34.1 (C-7), 35.9 (C-4), 36.9 (C-13), 38.1 (C-10), 39.6 (C-1), 40.8 (C-8), 41.8 (C-14), 45.5 (C-7′), 45.9 (C-19), 47.3 (C-17), 50.2 (С-18), 52.6 (C-2′, C-6′), 54.3 (C-5′), 54.5 (С-3′), 55.0 (C-9), 55.2 (С-5), 109.1 (C-29), 151.2 (C-20), 167.3 (C-3), 173.4 (C-28).

Synthesis of Compound 3

A solution of compound 2 (0.55 g, 1 mmol) in dioxane (30 mL) and 0.4 mL of SOCl₂ was stirred at room temperature for 30 minutes, then poured into H₂O (100 mL), the precipitate was filtered, washed, dried, and crystallized from EtOH.

3-Oxo-3а-Homo-3а-Aza-17-(4-Methylpiperazin-1-yl)-Carbonylo-Lup-20(29)-ene (3)

Yield 0.36 g, 87%, as a yellow solid.

R _f 0.10.

MP: 2270–229°С.

[α]_D ²⁰ +12.0° (c 1.00, CHCl₃);

Anal. Calcd. for С₃₅Н₅₇N₂O₂ (Мr 551.86): С 76.18; Н 10.41; N 7.61. Found: С 76.27; Н 10.52; N 7.71.

¹H NMR (300 MHz, CDCl₃): δ 0.89 (3H, s, H-25), 0.92 (3H, s, H-26), 1.01 (3H, s, H-24), 1.21 (3H, s, H-27), 1.29 (3H, s, H-23), 1.22-2.08 (21Н, m, H-1, H-2, H5-H7, H-9, H11-H13, H-15, H-16, H-18, H-21, H-22), 1.69 (3Н, s, H-30), 2.06-2.18 (4Н, m, H-3′, H-5′), 2.29 (3Н, s, H-7′), 2.43-2.55 (3Н, m, H-13, H-16), 2.68-3.20 (5Н, m, H-19, H-2′, H-6′), 4.59 (1H, s, H-29), 4.71 (1H, s, H-29), 6.13-6.22 (1H, m, NH);

¹³C NMR (CDCl₃, 75.5 MHz): δ 14.4 (C-27), 15.8 (C-25), 15.9 (C-26), 19.5 (C-11), 19.6 (C-30), 20.9 (C-6), 21.6 (C-24), 22.4 (C-23), 25.8 (C-12), 27.1 (C-2), 29.7 (C-15), 30.9 (C-21), 32.2 (C-16), 33.3 (C-22), 33.7 (C-7), 35.9 (C-4), 37.0 (C-13), 37.4 (C-10), 39.1 (C-1), 40.3 (C-8), 40.6 (C-14), 45.4 (C-7′), 45.9 (C-19), 47.3 (C-17), 51.1 (С-18), 52.3 (C-2′, C-6'), 53.7 (C-5'), 54.5 (С-3'), 55.0 (C-9), 56.9 (С-5), 109.7 (С-29), 150.4 (С-20), 173.8 (С-28), 176.7 (C-3).

Synthesis of Compound 4

LiAlH₄ (0.456 g; 12 mM) was added to a solution of compound 3 (0.55 g; 1 mM) in dry THF (30 mL) under a nitrogen atmosphere. The reaction was heated for 1 hour, then quenched with a solution of 1 M HCl and extracted with chloroform (3 × 60 mL). The combined organic layers were dried over CaCl₂, concentrated in vacuo and chromatographed on a Al₂O₃ column eluted with chloroform.

3-Deoxy-3а-Homo-3а-Aza-17-(4-Methylpiperazin-1-yl)-Carbonylolup-20(29)-ene (4)

Yield 0.46 g, 87%, as a white solid.

Rf 0.08.

MP: 153°С.

[α]_D ²⁰ +25° (c 1.00, CHCl₃).

Anal. Calcd. for С₃₅Н₅₉N₃O (Мr 537.88): C 78.16; Н 11.06; N 7.81. Found: С 78.24; Н 11.14; N 7.92.

¹H NMR (300 MHz, CDCl₃): δ 0.89 (3H, s, H-25), 0.92 (3H, s, H-26), 0.98 (3H, s, H-24), 1.02 (3H, s, H-27), 1.12 (3H, s, H-23), 1.17-2.08 (22Н, m, H-1, H-2, H5-H7, H-9, H11-H13, H-15, H-16, H-18, H-21, H-22), 1.69 (3Н, s, H-30), 2.29-2.45 (7Н, m, H-13, H-16, H-3′, H-5′), 2.32 (3Н, s, H-7′), 2.51-3.02 (6H, m, H-3, H-19, H-2′, H-6′), 3.03-3.21 (1H, m, NH), 4.54 (1H, s, H-29), 4.68 (1H, s, H-29).

¹³C NMR (CDCl₃, 75.5 MHz): δ 14.5 (C-27), 15.7 (C-25), 16.4 (C-26), 19.1 (C-11), 19.6 (C-30), 20.9 (C-6), 21.3 (C-24), 22.7 (C-23), 25.8 (C-12), 26.9 (C-2), 29.0 (C-15), 31.3 (C-21), 33.5 (C-16), 33.9 (C-22), 34.1 (C-7), 35.9 (C-3), 36.9 (C-13), 38.3 (C-10), 39.4 (C-1), 40.7 (C-8), 41.8 (C-14), 45.5 (C-7′), 45.9 (C-19), 47.3 (C-17), 50.2 (С-18), 52.4 (C-2′, C-6′), 54.3 (C-5′), 54.5 (С-3′), 55.0 (C-9), 56.9 (С-5), 63.2 (С-3), 109.8 (С-29), 150.2 (С-20), 174.1 (С-28).

Minimum Inhibitory Concentration

MIC in LB medium for BW25113 E. coli and BW25113 E. coli ΔtolC (JW5503 from Keio collection) was determined using broth microdilution assay. ²² The cell concentration was adjusted to approximately 5 × 105 cells/mL. The tested compounds were serially diluted twice in a 96-well 2 mL microplate (200 µL per well). Microplates were covered and incubated at 37°C with shaking. The OD600 of each well was measured, and the lowest concentration of the tested compound that resulted in no growth after 16–20 hours was assigned to the MIC value.

Testing the In Vivo Activity of 4

The reporter strain dtolC-pDualrep2 was used as previously described. ¹⁹ Briefly, 2 µL of compound 4 solutions 10 mg/mL were applied to an agar plate that already contained a lawn of the reporter strain. 10 mM erythromycin and 20 µM levofloxacin solutions were used as controls. After being incubated overnight at 37°C, the plate was scanned by ChemiDoc (Bio-Rad): “Cy3-blot” for RFP and “Cy5-blot” for Katushka2S.

Cytotoxicity Measurements

The measurements were carried out using the standard MTT method. ²³ 3000 cells per well for PC3, Huh7, and 22Rv1 cell lines or 4000 cells per well for VA13 and LNCAP were plated out in 135 µL of media in 96-well plate. DMEM-F12 media (Gibco) was used for VA13 and Huh7, and RPMI (Gibco) was used for PC3, 22Rv1 and LNCAP cell lines. Media was supplemented with 10% heat-inactivated FBS, 1 × Penstrep, 1 × Glutamax (Gibco). Cells were incubated in the 5% CO₂ incubator for first 16 hours without treating. Then 15 µL of media-dimethyl sulfoxide (DMSO) solutions of tested substances to the cells (final DMSO concentrations in the media were 1% or less) and treated cells 72 hours (triplicate each). The MTT reagent then was added to cells up to the final concentration of 0.5 g/L (10× stock solution in phosphate buffered saline was used) and incubated for 2 hours at 37°C in the incubator, under an atmosphere of 5% CO₂. The MTT solution was then discarded and 140 µL of DMSO was added. The plates were swayed on a shaker (80 rpm) to solubilize the formazan. The absorbance was measured using a microplate reader (VICTOR X5 Plate Reader) at a wavelength of 565 nm (in order to measure the formazan concentration). The results were used to construct a dose-response graph and to estimate CC₅₀ value by Prism software (GraphPad Software, Inc.).