Synthesis and biological activity of new 18β-glycyrrhetinic acid derivatives

Mohamed O. Radwan; Mohamed A.H. Ismail; Sahar El-Mekkawy; Nasser S.M. Ismail; Atef G. Hanna

doi:10.1016/j.arabjc.2013.06.032

View/Download PDF

Buy Reprints

PDF

Translate this page into:

Original Article

9 (

); 390-399

doi:

10.1016/j.arabjc.2013.06.032

Synthesis and biological activity of new 18β-glycyrrhetinic acid derivatives

Mohamed O. Radwan^{^a,⁎}, Mohamed A.H. Ismail^{^b}, Sahar El-Mekkawy^{^a}, Nasser S.M. Ismail^{^b}, Atef G. Hanna^{^a}

a Chemistry of Natural Compounds Department, National Research Centre, El-Behoos St. 33, Dokki-Cairo 12622, Egypt

b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, PB 11566, Abbasiya sq., Cairo, Egypt

⁎Corresponding author. Tel.: +20 1004991399; fax: +20 2 33370931. mohamedosman251@gmail.com (Mohamed O. Radwan)

Received: 2012-11-12, Accepted: 2013-6-26, Published: 2013-05-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

In an attempt to find out new, potent and safe anti-inflammatory molecules and as a contribution in the chemistry of triterpenes, a series of 18β-glycyrrhetinic acid (GTA) derivatives (4a–j, 5a–e, 7–9, 11–13) were synthesized and evaluated as anti-inflammatory agents using carrageenan induced rat paw edema method. The synthesized derivatives proved superior anti-inflammatory activity to GTA. Moreover, some of the produced derivatives demonstrated higher effect than prednisolone and indomethacin. This remarkable anti-inflammatory effect was combined with no detrimental effect on the gastrointestinal tract (GIT) of the test rats. All of the synthesized compounds were characterized by NMR spectroscopy and high-resolution ESI mass spectrometry.

Keywords

18β-Glycyrrhetinic acid

Anti-inflammatory

Show Related Articles from PubMed

1 Introduction

Typically possessing low toxicity and a broad spectrum of biological activities, plant triterpenoids are valuable raw materials for the creation of new drugs (Schopke and Hiller, 1990; Tolstikov et al., 1998; Platonov et al., 1995; Shon et al., 1998 ). Glycyrrhizic (GZA) and glycyrrhetinic acid (GTA), as well as their derivatives, exhibit a various biological effects, including anti-inflammatory and anti-ulcer activity (Tolstikov et al., 1997, 1998; Platonov et al., 1995; Shon et al., 1998 ). Consequently, they serve as pronounced starting materials for effective anti-inflammatory, anti-allergic and antiulcer preparations (Finney and Tarnoky, 1960; Gheorghiu et al., 1971; Yano et al., 1989 ). GZA, GTA and their derivatives affect the arachidonic acid cycle similar to the non-steroidal anti-inflammatory agents (Inoue et al., 1988). In vitro research has also demonstrated that GZA inhibits cyclooxygenase activity and prostaglandin formation (specifically prostaglandin E₂) (Ohuchi et al., 1981; Okimasu et al., 1983; Ohuchi and Tsurufuji, 1982 ). Moreover, GZA and GTA are known to inhibit phospholipase A₂ activity, an enzyme critical to numerous inflammatory processes (Kase et al., 1998).

Recently, some derivatives of GTA have shown their inhibitory activity against interleukin-1β (IL-1β)-induced prostaglandin E₂ (PGE₂) (Tsukahara et al., 2005). Also, GZA inhibits reactive oxygen species (ROS) generation by neutrophils. GZA significantly decreases neutrophil-generated O₂, H₂O₂ and OH in a dose-dependent manner. It is thought that one of GZA anti-inflammatory effects is attributed to this effect (Akamatsu et al., 1991; Wang and Nixon, 2001).

Some of GTA derivatives inhibited nitric oxide (NO) generation and suppressed superoxide anion formation by rat neutrophils; they also inhibited xanthine oxidase activity. These actions may have value in the therapeutical treatment or prevention of certain central as well as peripheral inflammatory diseases associated with the increase of (NO) production (Maitraie et al., 2009). Although being anti-inflammatory molecule, GTA is characterized by being anti-ulcer agent at the same time. Its anti-ulcer effect is attributed to the sequential inhibition of 15-hydroxyprostaglandin dehydrogenase and Δ¹³-ketoprostaglandin reductase; the two enzymes are involved in the gastric prostaglandin metabolism (Aly et al., 2005)^. Prostaglandins promote healing of ulcers by stimulating mucous secretion and cell proliferation in the stomach. Thus, the local increase of prostaglandin concentration by glycyrrhetinic-derived compounds, promotes healing of ulcers.

GTA produces an undesired aldosterone-like effect (Ulmann et al., 1975), potentiates the action of aldosterone (Ishikawa and Saito, 1980), and inhibits its metabolism. At the same time, GTA derivatives frequently exhibit a more pronounced therapeutic action than does the initial GTA and produce no aldosterone-like side effects Takahashi et al., 1980; Shibata et al., 1987). Hence, we aimed to make new derivatives that exceed GTA activity with a better safety profile.

2 Results and discussion

2.1

2.1 Chemistry

GTA was isolated from Glycyrrhiza glabra roots in our laboratory by an unpublished method. Its identity was confirmed by IR, m.p. [291–292 °C, lit. 292–294 (Abubakirov and Yatsyn, 1959)] and mixed m.p. The target derivatives were synthesized by modification of the two main active functional groups of GTA (20-COOH and 3-OH). For the synthesis of compounds 4 and 5 series, the hydroxyl group was protected by acetylation (Murav’ev and Savchenko, 1979) and the carboxylic group was activated by the preparation of acid chloride (3) using SOCl₂ (Adanin and Khaletskii, 1967) .The addition of few drops of dimethyl formamide (Ibrahim et al., 1995), to enhance the reaction led to decrease in the overall yield of the acid chloride. After purification, the acid chloride produced was reacted with some aryl, alicyclic, heterocyclic amines and aryl phenols to give the corresponding amides (4a–j) and esters (5a–e), respectively (Scheme 1). The acid chloride method resulted in a high yield for the produced amides and esters. The mixed anhydride of GTA was prepared by reacting GTA with ethylchloroformate in presence of triethylamine in dichloromethane, but it resulted in a poor yield for the produced amides (∼20%).

Compound 4g was hydrolyzed by alcoholic KOH to produce compound 6. The free –OH of the latter compound was esterified with each of succinic and phthalic anhydrides using excess amounts of both of them in presence of dry pyridine and molecular sieve 4A° to afford compounds 7 and 8, respectively (Tsukahara et al., 2005). Compound 9 was prepared through further amidation of compound 4g with p-toluidine via mixed anhydride formation (Scheme 2).

Methyl glycyrrhetate (10) was prepared by Fischer esterification of GTA with methanol; compounds 11–13 were prepared through the reaction of (10) with excess succinic, phthalic and maleic anhydrides, respectively in pyridine (Kondratenko et al., 2001) (Scheme 3). During the reaction with succinic and maleic anhydrides, reaction mixture got dark. It grows faster in case of maleic than succinic anhydride. Pyridine induces a chemical reaction of some violence which yields carbon dioxide and a black brittle residue (Rittenberg and Ponticorvo, 1960). Acetone was used during the working up to dissolve this black brittle residue.

2.2

2.2 Biological activity

In the present investigation, variable synthesized compounds were evaluated for their possible anti-inflammatory activity in a rat model of carrageenan-induced paw edema, which is a widely used animal model for determining the acute phase of inflammation. The anti-inflammatory potency of the tested compounds was compared with free GTA and two standard drugs, prednisolone and indomethacin. Figs. 1 and 2 demonstrate the potency of GTA and its synthesized derivatives versus prednisolone and indomethacin, respectively. It is noteworthy to mention that the derivatives 4g, 4h, 5a and 11 showed greater anti-inflammatory potency than both prednisolone and indomethacin. However, compounds 4i, 8 and 9 showed almost the same anti-inflammatory potency of the reference standards. On the other hand, free GTA showed the least potency as compared with the synthesized compounds.

Anti-inflammatory potency of GTA and its derivatives as compared with prednisolone.

Anti-inflammatory potency of GTA and its derivatives as compared with indomethacin.

At the end of the experiment, rats were killed under light ether anesthesia by cervical dislocation; their stomachs were excised, opened at the greater curvature and examined for the presence of ulcers. All rat groups that received GTA, its synthesized derivatives and prednisolone showed very few gastric lesions or no ulcers at all, compared to rat groups that received indomethacin, which showed some gastric ulcers with considerable severity.

3 Materials and methods

Melting points were uncorrected and determined with electrotherma capillary melting point apparatus. The NMR spectra were measured on a Varian Unity 300 (300.145 MHz) and on Buker AM-200 (200 MHz) spectrometers. ESI mass spectra were recorded on a Finnigan LCQ spectrometer with quaternary pump Rheos 4000 (Flux Instrument). Flash chromatography was carried out on silica gel (230–400 mesh). R_f values were measured on Polygram SIL G/UV₂₅₄ (Macherey–Nagel & Co.). Reaction progress was followed and monitored using thin layer chromatography (TLC, DF₂₅₄) and eluted with the following systems (a) benzene/ethyl acetate/AcOH (12:2:0.5) or (b) hexane/ethyl acetate (6:4). Visualization was carried out by either UV (254 nm, 365 nm) or spraying with methanol/sulfuric acid (5%) and then heated with air dryer.

3.1

3.1 Isolation of GTA from Glycyrrhiza glabra roots

The dry powdered root of Glycyrrhiza glabra (1 kg) was treated with 5 L of 5% sulfuric acid. The mixture was refluxed for 6 h and left to cool. The mixture was filtered off and the residue was carefully washed with water and dried. The residue was extracted by dry benzene in a soxhlet. The pale yellow extract was treated with 200 ml of acetic anhydride and the mixture was refluxed for 1 h, then it was left for 24 h at room temperature. The reaction mixture was poured on crushed ice, while stirring and the produced precipitate was filtered off and washed with water. The produced glycyrrhetinic acid acetate (2) was crystallized several times from methanol to produce colorless crystals [m.p. 315–317 °C, lit. 317–318 °C (Murav’ev and Savchenko, 1979)]. Compound 2 was saponified by refluxing with 5% alcoholic NaOH for 3 h to liberate free GTA (1).

3.2

3.2 Synthesis of the target derivatives

3.2.1

3.2.1 Glycyrrhetinic acid acetate (2), acetyl glycyrrhetyl chloride (3) and methyl glycyrrhetate (10)

The three compounds were prepared according to published methods (Murav’ev and Savchenko, 1979; Baltina et al., 1997).

3.2.2

3.2.2 General procedures for the preparation of compounds 4a–j and 5a–e

A solution of 3 (100 mg, 0.19 mmol) in dry benzene was added drop wise to a stirred solution of the appropriate amine or phenol (0.19 mmol) and triethylamine (27 μl, 0.19 mmol) in dry benzene or THF. After complete addition, the mixture was heated under reflux for 3 h and the course of reaction was monitored using TLC. At the end of the reaction, water was added; the mixture was washed with 2 N HCl (compounds 4a–j) or 2% NaOH (compounds 5a–e); benzene layer was separated, dried over anhydrous sodium sulfate and distilled off under vacuum. The formed amide or ester was purified by flash column chromatography using the appropriate solvent system.

3.2.3

3.2.3 N-(phenethyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4a)

M.p 121–123 °C; yield 68%; (+)-ESI-MS: m/z (%) 638 ([M + Na]⁺), 1254 ([2 M + Na]⁺) 614 ([M−H]⁻); (+)-HRESI-MS: m/z 638.41788 (calc: 638.41798 for C₄₀H₅₇NO₄Na); ¹H NMR: δ at 0.82 (s, 3H, CH₃-28), 0.86 (s, 6H, CH₃-23, 24), 1.07 (s, 3H, CH₃-27), 1.12 (s, 3H, CH₃-25), 1.25 (s, 3H, CH₃-29), 1.34 (s, 3H, CH₃-26), 2.01 (s, 3H, CH₃-32), 2.29 (s, 1H, CH-9), 2.78 (d, 1H, CH-1 eq), 2.85 (m, 2H, CH₂-34), 3.52 (m, 2H, CH₂-33), 4.47 (dd, 1H, CH-3), 5.45 (s, 1H, CH-12), 5.75 (t, 1H, NH), 7.22 (m, 5H, CH-2′, 3′, 4′, 5′, 6′); ¹³C NMR: δ at 38.74 (C-1), 23.68 (C-2), 80.56 (C-3), 37.97 (C-4), 54.95 (C-5), 17.30 (C-6), 32.61 (C-7), 45.28 (C-8), 61.64 (C-9), 36.86 (C-10), 199.85 (C-11), 128.33 (C-12), 169.09 (C-13), 43.52 (C-14), 26.34 (C-15), 26.31 (C-16), 31.78 (C-17), 47.87 (C-18), 41.02 (C-19), 43.11 (C-20), 31.34 (C-21), 37.34 (C-22), 27.98 (C-23), 16.61 (C-24), 16. 34 (C-25), 18.59 (C-26), 23.48 (C-27), 28.36 (C-28), 29.95 (C-29), 175.66 (C-30), 179.96 (C-31), 21.23 (C-32), 43.11 (C-33), 40.38 (C-34), 138.69 (C-1′), 128.64 (C-2′), 128.67 (C-3′), 126.57 (C-4′), 128.67 (C-5′), 128.64 (C-6′).

3.2.4

3.2.4 N-(4-chlorophenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4b)

M.p 281–283 °C; yield 82%; (+)-ESI-MS: m/z (%) 645 ([M + Na]⁺), 1267 ([2 M + Na]⁺), 621 ([M−H]⁻); (+)-HRESI-MS: m/z 623.3663 (calc: 623.3658 for C₃₈H₅₃NO₄Cl); ¹H NMR: δ at 0.83 (s, 3H, CH₃-28), 0.88 (s, 6H, CH₃-23, 24), 1.13 (s, 3H, CH₃-27), 1.14 (s, 3H, CH₃-25), 1.25 (s, 3H, CH₃-29), 1.39 (s, 3H, CH₃-26), 2.06 (s, 3H, CH₃-32), 2.37 (s, 1H, CH-9), 2.80 (d, 1H, CH-1 eq), 4.53 (dd, 1H, CH-3), 5.68 (1H, s, CH-12), 7.28 (d, 2H, CH-2’, 6’), 7.45 (d, 2H, CH-3′, 5′), 3 J (2′, 3′) = 3 J (5′, 6′) = 9 Hz; ¹³C NMR: δ at 38.77 (C-1), 23.52 (C-2), 80.57 (C-3), 38.01 (C-4), 54.99 (C-5), 17.34 (C-6), 32.68 (C-7), 45.37 (C-8), 61.76 (C-9), 36.91 (C-10), 199.91 (C-11), 128.56 (C-12), 168.88 (C-13), 44.56 (C-14), 26.38 (C-15), 26.38 (C-16), 32.01 (C-17), 48.16 (C-18), 41.82 (C-19), 43.20 (C-20), 31.62 (C-21), 37.37 (C-22), 28.02 (C-23), 16.66 (C-24), 16. 38 (C-25), 18.64 (C-26), 23.33 (C-27), 28.38 (C-28), 29.35 (C-29), 174.01 (C-30), 171.05 (C-31), 21.32 (C-32), 136.37 (C-1′), 121.34 (C-2′), 129.01 (C-3′), 128.77 (C-4′), 129.01 (C-5′), 121.34 (C-6′).

3.2.5

3.2.5 N-(benzyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4c)

M.p 134–136 °C; yield 67%; (+)-ESI-MS: m/z (%) 624 ([M + Na]⁺), 600 ([M−H]⁻); (+)-HRESI-MS: m/z 602.4203 (calc: 602.4203 for C₃₉H₅₆NO₄); ¹H NMR: δ at 0.76 (s, 3H, CH₃-28), 0.82 (s, 6H, CH₃-23, 24), 1.11 (s, 6H, CH₃-27, 25), 1.30 (s, 3H, CH₃-29), 1.36 (s, 3H, CH₃-26), 2.00 (s, 3H, CH₃-32), 2.28 (s, 1H, CH-9), 2.73 (d, 1H, CH-1 eq), 4.43 (s, 3H, CH₃ and CH₂-33), 5.51 (1H, s, CH-12), 5.91 (1H, s, NH), 7.24 (6H, m, CH-2′, 3′, 4′, 5′, 6′, CDCl₃); ¹³C NMR: δ at 38.74 (C-1), 23.48 (C-2), 80.56 (C-3), 37.99 (C-4), 54.95 (C-5), 17.32 (C-6), 32.56 (C-7), 45.31 (C-8), 61.66 (C-9), 36.86 (C-10), 199.95 (C-11), 128.37 (C-12), 169.07 (C-13), 43.60 (C-14), 26.34 (C-15), 26.34 (C-16), 31.89 (C-17), 48.12 (C-18), 41.81 (C-19), 43.15 (C-20), 31.43 (C-21), 37.40 (C-22), 28.00(C-23), 16.64 (C-24), 16. 35 (C-25), 18.63 (C-26), 23.22 (C-27), 28.39 (C-28), 29.52 (C-29), 175.53 (C-30), 173.03 (C-31), 21.29 (C-32), 43.60 (C-33), 138.57 (C-1′), 127.71 (C-2′), 128.76 (C-3′), 127.56 (C-4′), 128.76 (C-5′), 127.71 (C-6′).

3.2.6

3.2.6 N-(3-chloro-4-Methylphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4d)

M.p 167–169 °C; yield 73%; (+)-ESI-MS: m/z (%) 658 ([M + Na]⁺), 634([M−H]⁻); (+)-HRESI-MS: m/z 636.3800 (calc: 636.3814 for C₃₉H₅₅NO₄Cl), 658.3620 (calc: 658.3634 for C₃₉H₅₄NO₄ClNa); ¹H NMR: δ at 0.81 (s, 3H, CH₃-28), 0.87 (s, 6H, CH₃-23, 24), 1.12 (s, 6H, CH₃-27, 25), 1.24 (s, 3H, CH₃-29), 1.37 (s, 3H, CH₃-26), 2.05 (s, 3H, CH₃-32), 2.29 (s, 3H, CH₃-Ar), 2.36 (s, 1H, CH-9), 2.78 (d, 1H, CH-1 eq), 4.52 (dd, 1H, CH-3), 5.69 (s, 1H, CH-12), 7.11 (m, 1H, CH-5′), 7.29 (m, 1H, CH-6′), 7.58 (s, 1H, CH-2′), 7.71 (s, 1H, NH); ¹³C NMR: δ at 38.72 (C-1), 23.44 (C-2), 80.54 (C-3), 37.96 (C-4), 54.91 (C-5), 17.29(C-6), 32.62 (C-7), 45.45 (C-8), 61.68 (C-9), 36.84 (C-10), 200.19 (C-11), 128.35 (C-12), 169.55 (C-13), 43.21 (C-14), 26.32 (C-15), 26.32 (C-16), 31.94 (C-17), 48.26 (C-18), 41.47 (C-19), 43.21 (C-20), 31.51 (C-21), 37.33 (C-22), 27.99 (C-23), 16.63 (C-24), 16.31 (C-25), 18.56 (C-26), 23.33 (C-27), 28.35 (C-28), 29.56 (C-29), 174.16 (C-30), 171.08 (C-31), 21.29 (C-32),136.83 (C-1′), 118.31 (C-2′), 134.35 (C-3′), 130.84 (C-4′), 131.68 (C-5′), 120.64 (C-6′), 20.22 (CH₃- Ar).

3.2.7

3.2.7 N-(cyclohexyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4e)

M.p 201–203 °C; yield 77%; (+)-ESI-MS: m/z (%) 616 ([M + Na]⁺), 1210 ([2 M + Na]⁺); (+)-HRESI-MS: m/z 616.4333 (calc: 616.43362 for C₃₈H₅₉NO₄Na); ¹H NMR: δ at 0.77 (s, 3H, CH₃-28), 0.84 (s, 6H, CH₃-23, 24), 1.09 (s, 3H, CH₃-27), 1.12 (s, 3H, CH₃-25), 1.22 (s, 3H, CH₃-29), 1.36 (s, 3H, CH₃-26), 1.65 (m, 2H, CH₂-d), 2.02 (s, 3H, CH₃-32), 2.21 (m, 2H, CH-3′, 5′), 2.32 (s, 1H, CH-9), 2.76 (d, 1H, CH-1 eq), 3.00 (m, 2H, CH-2′, 6′), 4.48 (dd, 1H, CH-3), 5.65 (s, 1H, CH-12), 3.78 (m, 1H, CH-1′); ¹³C NMR: δ at 38.58 (C-1), 23.49 (C-2), 80.55 (C-3), 37.84 (C-4), 54.82 (C-5), 17.35 (C-6), 32.52 (C-7), 45.32 (C-8), 61.70 (C-9), 36.75 (C-10), 199.86 (C-11), 128.32 (C-12), 169.25 (C-13), 43.14 (C-14), 26.28 (C-15), 26.28 (C-16), 31.72 (C-17), 48.08 (C-18), 41.83 (C-19), 43.14 (C-20), 31.35 (C-21), 37.26 (C-22), 27.85 (C-23), 16.45 (C-24), 16. 20 (C-25), 18.61 (C-26), 23.30 (C-27), 28.50 (C-28), 28.51 (C-29), 174.51(C-30), 170.85 (C-31), 21.22 (C-32), 51.10 (C-1′), 33.28 (C-2′), 22.73 (C-3′), 28.49 (C-4′), 22.73 (C-5′), 33.28 (C-6′).

3.2.8

3.2.8 N-(4-Methylphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4f)

M.p 181–183 °C [lit. 180–182 °C 9 Dalimov et al., 2001)]; yield 69%; (+)-ESI-MS: m/z (%) 624 ([M + Na]⁺), 600 ([M−H]^-); (+)-HRESI-MS: m/z 624.4021 (calc: 624.4023 for C₃₉H₅₅NO₄Na); ¹H NMR: δ at 0.81 (s, 3H, CH₃-28), 0.84 (s, 6H, CH₃-23, 24), 1.14 (s, 3H, CH₃-27), 1.16 (s, 3H, CH₃-25), 1.24 (s, 3H, CH₃-29), 1.38 (s, 3H, CH₃-26), 2.01 (s, 3H, CH₃-32), 2.31 (s, 3H, CH₃-Ar), 2.38 (s, 1H, CH-9), 2.79 (d, 1H, CH-1 eq), 4.52 (dd, 1H, CH-3), 5.64 (s, 1H, CH-12), 7.10 (d, 2H, CH-3′, 5′), 7.38 (d, 2H, CH-2′, 6′), J_{2′, 3′} = J_{5′, 6′} = 6 Hz, 7.43 (s, 1H, NH); ¹C NMR: δ at 38.83 (C-1), 23.60 (C-2), 80.58 (C-3), 38.06 (C-4), 55.02 (C-5), 17.43 (C-6), 32.74 (C-7), 45.41 (C-8), 61.75 (C-9), 36.97 (C-10), 199.74 (C-11), 128.39 (C-12), 168.99 (C-13), 44.46 (C-14), 26.49 (C-15), 26.46 (C-16), 32.04 (C-17), 48.22 (C-18), 41.93 (C-19), 43.27 (C-20), 31.69 (C-21), 37.45 (C-22), 28.09 (C-23), 16.73 (C-24), 16. 43 (C-25), 18.73 (C-26), 23.39 (C-27), 28.44 (C-28), 29.42 (C-29), 173.72 (C-30), 170.86 (C-31), 21.36 (C-32), 135.19 (C-1′), 120.21 (C-2′), 129.36(C-3′), 133.87 (C-4′), 129.36 (C-5′), 120.21 (C-6′), 20.88 (CH₃- Ar).

3.2.9

3.2.9 N-(2-carboxyphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4g)

M.p 174–176 °C; yield 75%; (+)-ESI-MS: m/z (%) 654 ([M + Na]⁺), 630 ([M−H]⁻); (+)-HRESI-MS: m/z 654.3609 (calc: 654.3609 for C₃₉H₅₃NO₆Na); ¹H NMR: δ at 0.86 (s, 6H, CH₃-28, 24), 0.97 (s, 3H, CH₃-23), 1.18 (s, 3H, CH₃-27), 1.23 (s, 6H, CH₃-25, 29), 1.36 (s, 3H, CH₃-26), 2.04 (s, 3H, CH₃-32), 2.33 (s, 1H, CH-9), 2.65 (d, 1H, CH-1 eq), 4.49 (dd, 1H, CH-3), 5.94 (1H, s, CH-12), 6.60 (s, 1H, NH), 7.06 (t, 1H, CH-4′), 7.51(t, 1H, CH-5′), 8.03(d, 1H, CH-6′), 8.74 (d, 1H, CH-3′), 11.34 (br-s, 1H, Ar–COOH); ¹³C NMR: δ at 38.77 (C-1), 23.51 (C-2), 80.51 (C-3), 38.00 (C-4), 55.18 (C-5), 17.46 (C-6), 32.17 (C-7), 45.54 (C-8), 60.67 (C-9), 36.88 (C-10), 202.46 (C-11), 128.79 (C-12), 170.69 (C-13), 45.54 (C-14), 26.18 (C-15), 25.50 (C-16), 31.92 (C-17), 46.52 (C-18), 43.87 (C-19), 43.87 (C-20), 30.38 (C-21), 37.10 (C-22), 28.05 (C-23), 16.79 (C-24), 14. 11 (C-25), 18.48 (C-26), 22.68 (C-27), 29.35 (C-28), 29.69 (C-29), 175.97 (C-30), 170.99 (C-31), 21.30 (C-32), 141.82 (C-1′), 115.67 (C-2′), 131.23 (C-3′), 122.52 (C-4′), 134.48 (C-5′), 120.54 (C-6′), 164.54 (Ar-COOH).

3.2.10

3.2.10 N-(5-Methyl isoxazol 3-yl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4h)

M.p 255–257 °C; yield 71%; (+)-ESI-MS: m/z (%) 615 ([M + Na]⁺), 591([M−H]⁻); (+)-HRESI-MS: m/z 615.3767 (calc: 615.3768 for C₃₆H₅₂N₂O₅Na); ¹H NMR: δ at 0.77 (s, 3H, CH₃-28), 0.84 (s, 6H, CH₃-24, 23), 1.08 (s, 3H, CH₃-27), 1.12 (s, 3H, CH₃-25), 1.21 (s, 3H, CH₃-29), 1.35 (s, 3H, CH₃-26), 2.02 (s, 3H, CH₃-32), 2.32 (s, 1H, CH-9), 2,36 (s, 3H, CH₃-33), 2.78 (d, 1H, CH-1 eq), 4.48 (dd, 1H, CH-3), 5.68 (s, 1H, CH-12), 6.74 (s, 1H, CH-4’), 9.06 (s, 1H, NH); ¹³C NMR: δ at 38.77 (C-1), 23.52 (C-2), 80.59 (C-3), 38.00 (C-4), 55.00 (C-5), 17.34 (C-6), 32.68 (C-7), 45.32 (C-8), 61.67 (C-9), 36.88 (C-10), 199.75 (C-11), 128.65 (C-12), 169.70 (C-13), 44.58 (C-14), 26.38 (C-15), 26.35 (C-16), 31.68 (C-17), 47.66 (C-18), 41.06 (C-19), 43.13 (C-20), 32.23 (C-21), 37.44 (C-22), 28.01 (C-23), 16.64 (C-24), 16. 34 (C-25), 18.63 (C-26), 23.33 (C-27), 28.34 (C-28), 29.64 (C-29), 174.60 (C-30), 170.97 (C-31), 21.26 (C-32), 158.54 (C-3′), 96.92 (C-4′), 168.57 (C-5′), 12.57 (CH₃-isoxazole).

3.2.11

3.2.11 N-[2-(4-sulfamoylphenyl)ethyl]-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4i)

M.p 153–155 °C; yield 78%; (+)-ESI-MS: m/z (%) 717 ([M + Na]⁺), 1412 ([2 M + Na]⁺); (+)-HRESI-MS: m/z 717.3913 (calc: 717.3908 for C₄₀H₅₈N₂O₆SNa); ¹H NMR: δ at 0.81 (s, 3H, CH₃-28), 0.83 (s, 6H, CH₃-23, 24), 1.04 (s, 3H, CH₃-27), 1.09 (s, 3H, CH₃-25), 1.10 (s, 3H, CH₃-29), 1.27 (s, 3H, CH₃-26), 2.02 (s, 3H, CH₃-32), 2.26 (s, 1H, CH-9), 2.65 (d, 1H, CH-1 eq), 2.9 (m, 2H, CH₂-34), 3.76 (m, 2H, CH₂-33), 4.45 (dd, 1H, CH-3), 5.02 (s, 1H, CH-12), 5.82 (t, 1H, NH), 7.29 (d, 2H, CH-3′, 5′), 7.81 (d, 2H, CH-2′, 6′), J_2′,3′ = J_{5′, 6′} = 6 Hz; ¹³C NMR: δ at 38.57 (C-1), 23.46 (C-2), 80.52 (C-3), 37.98 (C-4), 54.95 (C-5), 17.27 (C-6), 32.60 (C-7), 45.49 (C-8), 61.77 (C-9), 36.94 (C-10), 201.24 (C-11), 127.99 (C-12), 170.55 (C-13), 43.76 (C-14), 26.33 (C-15), 26.23 (C-16), 31.90 (C-17), 48.09 (C-18), 41.53 (C-19), 43.27 (C-20), 31.55 (C-21), 37.52 (C-22), 27.97 (C-23), 16.61 (C-24), 16. 37 (C-25), 18.63 (C-26), 23.19 (C-27), 28.51 (C-28), 29.53 (C-29), 176.00 (C-30), 171.07 (C-31), 21.27 (C-32), 40.59 (C-33), 35.51 (C-34), 144.33 (C-1′), 129.50 (C-2′), 126.79 (C-3′), 140.94 (C-4′), 126.79 (C-5′), 129.50 (C-6′).

3.2.12

3.2.12 N-(morpholyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4j)

M.p 169–171 °C; yield 75%; (+)-ESI MS: m/z (%) 604 ([M + Na]⁺, 100), 1185 ([2 M + Na]⁺, 40), 580 ([M−H]⁻; (+)-HRESI MS: m/z 582.4151 (calc: 582.4153 for C₃₆H₅₆NO₅), 604.3973 (calc: 604.3972 for C₃₆H₅₅NO₅Na); ¹H NMR: δ at 0.79 (s, 3H, CH₃-28), 0.82 (s, 6H, CH₃-23, 24), 1.07 (s, 3H, CH₃-27), 1.10 (s, 3H, CH₃-25), 1.19 (s, 3H, CH₃-29), 1.31 (s, 3H, CH₃-26), 2.02 (s, 3H, CH₃-32), 2.17 (m, 4H, CH₂-2′, 6′), 2.31 (s, 1H, CH-9), 2.77 (d, 1H, CH-1 eq), 2.98 (m, 4H, CH₂-3′, 5′), 4.47 (dd, 1H, CH-3), 5.63 (s, 1H, CH-12); ¹³C NMR: δ at 38.45 (C-1), 23.23 (C-2), 80.25 (C-3), 37.69 (C-4), 54.63 (C-5), 17.04 (C-6), 32.70 (C-7), 45.64 (C-8), 61.34 (C-9), 36.61 (C-10), 199.64 (C-11), 128.10 (C-12), 169.42 (C-13), 44.96 (C-14), 26.37 (C-15), 26.07 (C-16), 32.38 (C-17), 47.81 (C-18), 42.98 (C-19), 43.48 (C-20), 31.43 (C-21), 37.38 (C-22), 26.59 (C-23), 16.73 (C-24), 16. 09 (C-25), 18.36 (C-26), 22.76 (C-27), 27.72 (C-28), 28.11 (C-29), 173.76 (C-30), 170.62 (C-31), 20.98 (C-32), 22.43 (C-2′), 50.83 (C-3′), 50.83 (C-5′), 22.43 (C-6′).

3.2.13

3.2.13 (3,5-Dimethylphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5a)

M.p 207–209 °C; yield 69%; (+)-ESI-MS: m/z (%) 617([M + H]⁺), 639 ([M + Na]⁺), 1455 ([2 M + Na]⁺); (+)-HRESI-MS: m/z 617.4171 (calc: 617.4201 for C₄₀H₅₇O₅); ¹H NMR: δ at 0.87 (s, 9H, CH₃-28, 23, 24), 1.15 (s, 6H, CH₃-27, 25), 1.25 (s, 3H, CH₃-25), 1.33 (s, 3H, CH₃-29), 1.39 (s, 3H, CH₃-26), 2.05 (s, 3H, CH₃-32), 2.31 (s, 1H, CH-9), 2.32 (s, 6H, two CH₃–Ar), 2.79 (d, 1H, CH-1 eq), 4.52 (dd, 1H, CH-3), 5.69 (s, 1H, CH-12), 6.64 (s, 2H, CH-2’, 6’), 6.86 (s, 1H, CH-4′); ¹³C NMR: δ at 39.81 (C-1), 23.99 (C-2), 81.05 (C-3), 38.48 (C-4), 55.44 (C-5), 17.81 (C-6), 33.14 (C-7), 45.85 (C-8), 62.16 (C-9), 37.34 (C-10), 199.92 (C-11), 128.00 (C-12), 169.51 (C-13), 44.66 (C-14), 26.86 (C-15), 26.86 (C-16), 31.58 (C-17), 48.85 (C-18), 41.49 (C-19), 43.29 (C-20), 30.61 (C-21), 38.17 (C-22), 28.48 (C-23), 17.12 (C-24), 16. 15 (C-25), 19.10 (C-26), 23.80 (C-27), 28.61 (C-28), 29.03 (C-29), 177.80 (C-30), 171.76 (C-31), 21.79 (C-32), 150.65 (C-33), 119.40 (C-34), 139.78 (C-1′), 129.07 (C-2′), 139.78 (C-3′), 119.40 (C-4′), 39.81 (C-5′), 32.99 (C-6′), 21.69 (Two CH₃–Ar).

3.2.14

3.2.14 (4-methylphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5b)

M.p 221–223 °C; yield 71%; (+)-ESI-MS: m/z (%) 625 ([M + Na]⁺), 601 ([M−H]⁻); (+)-HRESI-MS: m/z 603.4043 (calc: 603.4044 for C₃₉H₅₅O₅); ¹H NMR: δ at 0.82 (s, 3H, CH₃-28), 0.85 (s, 6H, CH₃-23, 24), 1.14 (s, 3H, CH₃-27), 1.15 (s, 3H, CH₃-25), 1.33 (s, 3H, CH₃-29), 1.37 (s, 3H, CH₃-26), 2.02 (s, 3H, CH₃-32), 2.35 (s, 3H, CH₃-Ar), 2.37 (s, 1H, CH-9), 2.79 (d, 1H, CH-1 eq), 4.53 (dd, 1H, CH-3), 5.63 (s, 1H, CH-12), 6.9 (d, 2H, CH-3′, 5′), 7.17 (d, 2H, CH-2′, 6′), J_{2′, 3′} = J_{5′, 6′} = 6 Hz; ¹³C NMR: δ at 38.78 (C-1), 23.61 (C-2), 80.56 (C-3), 38.06 (C-4), 55.02 (C-5), 17.43 (C-6), 32.74 (C-7), 45.40 (C-8), 61.71 (C-9), 36.96 (C-10), 199.76 (C-11), 128.50 (C-12), 168.74 (C-13), 44.21 (C-14), 26.50 (C-15), 26.47 (C-16), 31.95 (C-17), 48.46 (C-18), 41.13 (C-19), 43.23 (C-20), 31.20 (C-21), 37.76 (C-22), 28.08 (C-23), 16.73 (C-24), 16. 44 (C-25), 18.74 (C-26), 23.39 (C-27), 28.14 (C-28), 29.62 (C-29), 175.03 (C-30), 170.71 (C-31), 21.34 (C-32), 148.39 (C-33), 120.98 (C-34), 129.81 (C-1′), 135.26 (C-2′), 129.81 (C-3′), 120.98 (C-4′), 38.78 (C-5′), 23.61 (C-6′), 20.90 (CH₃- Ar).

3.2.15

3.2.15 (4-ethyl carboxyphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5c)

M.p 209–211 °C; yield 76%; (+)-ESI-MS: m/z (%) 683 ([M + Na]⁺); (+)-HRESI-MS: m/z 683.3919 (calc: 683.3918 for C₄₁H₅₆O₇Na); ¹H NMR: δ at 0.85 (s, 9H, CH₃-28, 23, 24), 1.12 (s, 6H, CH₃-27, 25), 1.36 (s, 9H, CH₃-26, 29), 1.42 (t, 3H, O–CH₂–CH₃), 2.02 (s, 3H, CH₃-32), 2.34 (s, 1H, CH-9), 2.76 (d, 1H, CH-1 eq), 4.35 (q, 2H, O⚌C–O–CH₂), 4.49 (dd, 1H, CH-3), 5.65 (s, 1H, CH-12), 7.08 (d, 2H, CH-3′, 5′), 8.06 (d, 2H, CH-2′, 6′), J_{2′, 3′} = J_{5′, 6′} = 9 Hz; ¹³C NMR: δ at 38.72 (C-1), 23.52 (C-2), 80.56 (C-3), 38.02 (C-4), 54.98 (C-5), 17.34 (C-6), 32.67 (C-7), 45.38 (C-8), 61.72 (C-9), 36.90 (C-10), 200.02 (C-11), 128.64 (C-12), 168.74 (C-13), 44.41 (C-14), 26.39 (C-15), 26.39 (C-16), 31.93 (C-17), 48.45 (C-18), 40.97 (C-19), 43.18 (C-20), 31.08 (C-21), 37.72 (C-22), 28.05 (C-23), 16.66 (C-24), 16. 38 (C-25), 18.64 (C-26), 23.36 (C-27), 28.05 (C-28), 28.57 (C-29), 174.61 (C-30), 171.04 (C-31), 21.32 (C-32), 154.40 (C-33), 121.43 (C-34), 131.14 (C-1′), 134.67 (C-2′), 131.14 (C-3′), 121.43 (C-4′), 38.72 (C-5′), 23.52 (C-6′), 167.90 (O⚌C–Ar), 61.08 (O–CH₂), 14.31(CH₂–CH₃).

3.2.16

3.2.16 (4-chlorophenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5d)

M.p 241–243 °C; yield 78%; (+)-ESI-MS: m/z (%) 645 ([M + Na]⁺), 621 ([M−H]⁻); (+)-HRESI-MS: m/z 623.3496 (calc: 623.3497 for C₃₈H₅₂O₅Cl); ¹H NMR: δ at 0.87 (s, 9H, CH₃-28, 23, 24), 1.15 (s, 6H, CH₃-27, 25), 1.33 (s, 3H, CH₃-29), 1.38 (s, 3H, CH₃-26), 2.04 (s, 3H, CH₃-32), 2.36 (s, 1H, CH-9), 2.78 (d, 1H, CH-1 eq), 4.51 (dd, 1H, CH-3), 5.66 (s, 1H, CH-12), 7.02 (d, 2H, CH-2′, 6′), 7.34 (d, 2H, CH-3′, 5′),J_{2′, 3′} = J_{5′, 6′} = 9 Hz; ¹³C NMR: δ at 38.71 (C-1), 23.51 (C-2), 80.55 (C-3), 38.00 (C-4), 54.95 (C-5), 17.32 (C-6), 32.65 (C-7), 45.37 (C-8), 61.70 (C-9), 36.88 (C-10), 200.04 (C-11), 128.61 (C-12), 168.78 (C-13), 44.28 (C-14), 26.36 (C-15), 26.36 (C-16), 31.90 (C-17), 48.47 (C-18), 40.97 (C-19), 43.16 (C-20), 31.06 (C-21), 37.68 (C-22), 28.02 (C-23), 16.64 (C-24), 16. 37 (C-25), 18.62 (C-26), 23.33 (C-27), 28.02 (C-28), 28.55 (C-29), 174.88 (C-30), 171.03 (C-31), 21.30 (C-32), 149.50 (C-33), 122.81 (C-34), 129.49 (C-1′), 131.10 (C-2′), 129.49 (C-3′), 122.81 (C-4′), 38.71 (C-5′), 23.51 (C-6′).

3.2.17

3.2.17 (4-bromophenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5e)

M.p 243–245 °C; yield 79%; (+)-ESI-MS: m/z (%) 667 ([M + H]⁺); (+)-HRESI-MS: m/z 667.2989 (calc: 667.2992 for C₃₈H₅₂O₅Br).¹H NMR: δ at 0.87 (s, 9H, CH₃-28, 23, 24), 1.15 (s, 6H, CH₃-27, 25), 1.33 (s, 3H, CH₃-29), 1.36 (s, 3H, CH₃-26), 2.05 (s, 3H, CH₃-32), 2.36 (s, 1H, CH-9), 2.78 (d, 1H, CH-1 eq), 4.52 (dd, 1H, CH-3), 5.66 (s, 1H, CH-12), 6.93 (d, 2H, CH-2′, 6′), 7.49 (d, 2H, CH-3′, 5′), J_{2′, 3′} = J_{5′, 6′} = 9 Hz; ¹³C NMR: δ at 38.71 (C-1), 23.52 (C-2), 80.00 (C-3), 38.00 (C-4), 54.96 (C-5), 17.32 (C-6), 32.65 (C-7), 45.37 (C-8), 61.70 (C-9), 36.87 (C-10), 200.03 (C-11), 128.62 (C-12), 168.76 (C-13), 44.30 (C-14), 26.38 (C-15), 26.38 (C-16), 31.90 (C-17), 48.46 (C-18), 40.96 (C-19), 43.16 (C-20), 31.06 (C-21), 37.68 (C-22), 28.02 (C-23), 16.65 (C-24), 16. 37 (C-25), 18.63 (C-26), 23.33 (C-27), 28.02 (C-28), 28.55 (C-29), 174.62 (C-30), 171.03 (C-31), 21.31 (C-32), 149.78 (C-33), 123.25 (C-34), 132.47 (C-1′), 119.90 (C-2′), 132.47 (C-3′), 123.25 (C-4′), 38.71 (C-5′), 23.52 (C-6′).

3.2.18

3.2.18 Preparation of N-(2-carboxyphenyl)-3β-hydroxy-11-ketoolean-12-en-18-βH-30-amide (6)

To a solution of 4g (500 mg, 0.75 mmol) in MeOH (50 ml) was added KOH (490 mg). After being refluxed for 3 h, the reaction mixture was neutralized with 2 N HCl, the formed precipitate was filtered under vacuum, washed with water and dried to give a yellowish residue. It was crystallized several times from methanol to give rise to 360 mg of compound 6.

m.p 279–280 °C; yield: 77%; ¹H NMR: δ at0.78 (s, 3H, CH₃-28), 0.86 (s, 3H, CH₃-24), 0.89 (s, 3H, CH₃-23), 1.08 (s, 3H, CH₃-27), 1.12 (s, 3H, CH₃-25), 1.25 (s, 3H, CH₃-29), 1.39 (s, 3H, CH₃-26), 2.40 (s, 1H, CH-9), 2.75 (d, 1H, CH-1 eq), 4.52 (dd, 1H, CH-3), 5.97 (s, 1H, CH-12), 7.09(t, 1H, CH-4′), 7.56(t, 1H, CH-5’), 8.10(d, 1H, CH-6′), 8.76 (d, 1H, CH-3′), 11.63 (br-s, 1H, Ar-COOH).

3.2.19

3.2.19 General procedures for the preparation of compounds 7 and 8

To a solution of 6 (150 mg, 0.26 mmol) in dry pyridine (10 ml), was added succinic anhydride (400 mg, 4 mmol) for the preparation of compound 7 or phthalic anhydride (600 mg, 4 mmol) for the preparation of compound 8, in presence of 4A° molecular sieve. The mixture was refluxed for 8 h, neutralized by hydrochloric acid. The formed precipitate was filtered under vacuum, washed with water and dried. Each of the residues was purified on silica gel column chromatography.

3.2.20

3.2.20 N-(2-carboxyphenyl)-3β-O-carboxypropanoyloxy-11-oxoolean-18β-H-30-amide (7)

m.p 169–170 °C; yield:59%; (+)-ESI-MS: m/z (%) 688 ([M-H]^-, 100); (+)-HRESI-MS: m/z 688.3851 (calc: 688.3855 for C₄₁H₅₄NO₈). ¹H NMR: δ at 0.77 (s, 3H, CH₃-28), 0.83 (s, 3H, CH₃-24), 0.86 (s, 3H, CH₃-23), 1.08 (s, 3H, CH₃-27), 1.11 (s, 3H, CH₃-25), 1.23 (s, 3H, CH₃-29), 1.39 (s, 3H, CH₃-26), 2.39 (s, 1H, CH-9), 2.66 (t, 4H, CH₂-32, 33),2.75 (d, 1H, CH-1 eq), 4.52 (dd, 1H, CH-3), 5.97 (s, 1H, CH-12), 7.08(t, 1H, CH-4′), 7.54(t, 1H, CH-5′), 8.09(d, 1H, CH-6′), 8.78 (d, 1H, CH-3’), 11.63 (br-s, 1H, Ar–COOH); ¹³C NMR: δ at 38.87 (C-1), 23.45 (C-2), 81.04 (C-3), 38.05 (C-4), 54.99 (C-5), 17.30 (C-6), 32.63 (C-7), 45.75 (C-8), 61.71 (C-9), 37.08 (C-10), 201.80 (C-11), 128.79 (C-12), 170.72 (C-13), 45.52 (C-14), 26.45 (C-15), 26.35 (C-16), 31.89 (C-17), 47.95 (C-18), 41.04 (C-19), 43.40 (C-20), 31.02 (C-21), 37.64 (C-22), 28.01 (C-23), 16.70 (C-24), 16. 43 (C-25), 18.72 (C-26), 23.35 (C-27), 28.50 (C-28), 29.03 (C-29), 177.12 (C-30), 175.25 (C-31), 29.69 (C-32), 29.69 (C-33), 172.14 (C-34), 142.23 (C-1′), 114.95 (C-2′), 131.54 (C-3′), 122.46 (C-4′), 134.90 (C-5′), 120.54 (C-6′), 171.82 (Ar–COOH).

3.2.21

3.2.21 N-(2-carboxyphenyl)-3β-O-phthaloyl-11-oxoolean-18β-H-30-amide (8)

m.p 177–178 °C; yield:62%; (+)-ESI-MS: m/z (%) 760 ([M + Na]⁺), 736 ([M−H]⁻); (+)-HRESI-MS: m/z 760.3813 (calc: 760.3820 for C₄₅H₅₅NO₈Na). ¹H NMR: δ at 0.77 (s, 3H, CH₃-28), 0.90 (s, 3H, CH₃-24), 0.96 (s, 3H, CH₃-23), 1.09 (s, 3H, CH₃-27), 1.10 (s, 3H, CH₃-25), 1.28 (s, 3H, CH₃-29), 1.41 (s, 3H, CH₃-26), 2.43 (s, 1H, CH-9), 2.78 (d, 1H, CH-1 eq), 4.79 (dd, 1H, CH-3), 6.13 (s, 1H, CH-12), 7.06 (t, 1H, CH-4′′), 7.52 (m, 3H, CH-5′, 4′, 5′), 7.67 (m, 1H, CH-6′),7.86 (s, 1H,CH-3′′), 8.03 (d, 1H, 6′), 8.76 (d, 1H, 3′).

3.2.22

3.2.22 Preparation of N-[2-(N-P-tolyl-benzamide)]-phenyl-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (9)

Compound 4g (100 mg, 0.15 mmol) was dissolved in 10 ml dry dichloromethane and triethylamine (25 μl, 0.15 mmol) was added. After stirring for 5 min, 0.02 ml of ethylchloroformate was added, followed by stirring for one hour, then 17 mg (0.15 mmol) of p-toluidine was added. Stirring is continued over night. Water (10 ml) was added and the organic layer was separated. It was washed at first with 2 N HCl followed with water, dried over anhydrous sodium sulfate and the solvent distilled off. The residue was purified on silica gel column chromatography.

m.p 149–150 °C; yield: 67%; (+)-ESI-MS: m/z (%) 643 ([M + Na]⁺, 100), 719 ([M−H]⁻); (+)-HRESI-MS: m/z 743.4382 (calc: 743.4394 for C₄₆H₆₀N₂O₅Na). ¹H NMR: δ at 0.77 (s, 3H, CH₃-28), 0.85 (s, 6H, CH₃-23, 24), 1.08 (s, 3H, CH₃-27), 1.13 (s, 3H, CH₃-25), 1.20 (s, 3H, CH₃-29), 1.37 (s, 3H, CH₃-26) 2.03 (s, 3H, CH₃-32), 2.29 (s, 3H, CH₃-Ar), 2.34 (s, 1H, CH-9), 2.76 (d, 1H, CH-1 eq), 4.50 (dd, 1H, CH-3), 5.79 (1H, s, CH-12), 7.07(t, 1H, CH-4′), 7.12 (d, 2H, CH-3″, 5″), 7.42 (d, 2H, CH-2″, 6″), 7.51 (t, 1H, CH-5′), 7.62 (d, 1H, CH-6′),8.15 (s, 1H, NH), 8.57 (d, 1H, CH-3′), 11.26 (s, 1H, NH).

3.2.23

3.2.23 General procedures for the preparation of compounds 11–13

A mixture of 100 mg (0.19 mmol) of compound 10 and 0.8 mmol of the appropriate acid anhydride were boiled for 10 h in 2 ml dry pyridine, in presence of 200 mg 4A° molecular sieve, without access of water. The reaction mixture was diluted with 2 ml of acetone, acidified with hydrochloric acid to pH ∼ 3–4. The residue was filtered under vacuum, washed with hot water, dried over anhydrous sodium sulfate and the solvent distilled off. Each residue was purified on silica gel column chromatography. It was eluted with hexane–ethyl acetate system, (step gradient mode, 8:2–7:3 v/v).

3.2.24

3.2.24 3-O-β-carboxypropionyl-11-oxoolean-12-en-18β-H-20β-O-methyl ester (11)

m.p 262–265 °C [lit. 262–264 °C (Kondratenko et al., 2001)]; yield: 68%;(+)-ESI-MS: m/z (%) 607 ([M + Na]⁺), 583 ([M-H]^-); (+)-HRESI-MS: m/z 583.3646 (calc: 583.3640 for C₃₅H₅₁O₇).¹H NMR: δ at 0.78 (s, 3H, CH₃-28), 0.85 (s, 3H, CH₃-24, 23), 1.10 (s, 3H, CH₃-27), 1.12 (s, 3H, CH₃-25), 1.23 (s, 3H, CH₃-29), 1.34 (s, 3H, CH₃-26), 2.33 (s, 1H, CH-9), 2.63 (m, 4H, CH₂-32, 33), 2.77 (d, 1H, CH-1 eq), 3.66 (s, 3H, CH₃-35), 4.52 (dd, 1H, CH-3),5.64 (s, 1H, CH-12).

3.2.25

3.2.25 3-O-β-phthaloyl-11-oxoolean-12-en-18β-H-20β-O-methyl ester (12)

m.p 156–158 °C(lit. 154–157 °C (Kondratenko et al., 2001); yield: 71%;(+)-ESI-MS: m/z (%) 655 ([M + Na]⁺), 631 ([M−H]⁻); (+)-HRESI-MS: m/z 631.3642 (calc: 631.3640 for C₃₉H₅₁O₇). ¹H NMR: δ at 0.78 (s, 3H, CH₃-28), 0.92 (s, 3H, CH₃-24), 0.96 (s, 3H, CH₃-23), 1.11 (s, 3H, CH₃-27), 1.13 (s, 3H, CH₃-25), 1.23 (s, 3H, CH₃-29), 1.36 (s, 3H, CH₃-26), 2.37 (s, 1H, CH-9), 2.85 (d, 1H, CH-1 eq), 3.67 (s, 3H, CH₃-32), 4.79 (dd, 1H, CH-3), 5.67 (s, 1H, CH-12), 7.56 (m, 2H, CH-4′, 5′), 7.75 (m, 1H, CH-6′), 7.93 (m, 1H, CH-3′).

3.2.26

3.2.26 3-O-β-Carboxy-trans-propenoyl-11-oxoolean-12-en-18β-H-20β-O-methyl ester (13)

m.p 209–211 °C; yield: 45%;(+)-ESI-MS: m/z (%) 605 ([M + Na]⁺), 581 ([M-H]^-); (+)-HRESI-MS: m/z 581.3485 (calc: 581.3484 for C₃₅H₄₉NO₇). ¹H NMR: δ at 0.78 (s, 3H, CH₃-28), 0.87 (s, 3H, CH₃-24), 0.90 (s, 3H, CH₃-23), 1.11 (s, 3H, CH₃-27), 1.13 (s, 3H, CH₃-25), 1.23 (s, 3H, CH₃-29), 1.35 (s, 3H, CH₃-26), 2.35 (s, 1H, CH-9), 2.80 (d, 1H, CH-1 eq), 3.67 (s, 3H, CH₃-35), 4.60 (dd, 1H, CH-3), 5.65 (s, 1H, CH-12), 6.82 (s, 2H, CH-32, 33).

3.3

3.3 Anti-inflammatory testing

Rats used in the biological testing of glycyrrhetinic acid and its derivatives were obtained from The Animal House Colony of the National Research Centre (NRC), Egypt. Seventy mature Wistar rats of both sexes, weighing 150–200 g were used. All animals were housed in hygienic cages in well ventilated rooms with exhaust fans; received standard pellet diet and water were provided ad libitum. The study was performed at the Pharmacology Research Unit- NRC and was approved by the Ethics Committee of The National Research Centre and in accordance with the recommendations of the proper care and use of laboratory animals (published by the National Academy of Science, National Academy Press, Washington, D.C.).

The anti-inflammatory test was performed according to the method of (Winter et al., 1962). Paw edema was induced in rats by subcutaneous (s.c.) injection of 0.1 ml of 1% (w/v) carageenan in distilled water in the sub-plantar region of their left hind paws. A group of rats was left without any treatment but were given a respective volume of the solvent (few drops of tween-80 in distilled water), and was kept as a control group. Drugs were administered orally at a dose of 100 mg/kg, one hour before carageenan injection. Oral prednisolone (Hostacortin-H®, 5 mg/kg) and indomethacin (Indocid®, 20 mg/kg) were administered to two groups of rats as reference drugs. The paw volume of each rat was measured using fluid displacement method, utilizing plethysmometer apparatus at 0–4 h of carageenan injection.

Edema rate and inhibition rate of each group were calculated at the previously mentioned time intervals as follows:

(1)

Edema rate (%) = Vt - Vo / Vo Inhibition rate (%) = Ec - Et / Ec

where:

Vo is the volume before carageenan injection (ml); Vt is the volume at t hour after carageenan injection (ml);

Ec is the edema rate of the control group; Et is the edema rate of treated group.

The anti-inflammatory potencies of glycyrrhetinic acid and its derivatives were calculated by comparing their inhibition rate at different time intervals; with those obtained from animals receiving either prednisolone or indomethacin, respectively, as standard anti-inflammatory drugs. Statistical analysis of the results, was done using analytical software named SPSS statistics 17.0, Release (Aug. 23, 2008), Chicago, USA. (See Tables 1 and 2).

Table 1 Anti-inflammatory potency of glycyrrhetic acid and some of its derivatives in comparison to prednisolone.

Compound	Anti-inflammatory potency (%)				Average potency in 4 h (%)
Compound	1 h	2 h	3 h	4 h	Average potency in 4 h (%)
4g	153.3	131.7	130.9	139.1	138.8
4h	149.9	121.6	123.6	139.1	133.6
4i	111.5	105.8	94.2	94.8	101.6
5a	102.0	135.4	131.7	134.5	125.9
7	101.2	71.6	64.3	65.3	75.6
8	78.7	84.9	106.9	101.2	92.9
9	100.0	98.1	99.4	98.1	98.9
11	168.9	125.7	111.7	119.5	131.5
12	89.0	75.4	66.2	68.0	74.6
13	66.3	55.8	54.1	56.1	58.1
GTA	58.4	46.5	43.6	43.5	48.0
Prednisolone	100.0	100.0	100.0	100.0	100.0

Table 2 Anti-inflammatory potency of glycyrrhetic acid and some of its derivatives in comparison to indomethacin.

Compound	Anti-inflammatory potency (%)				Average potency in 4 h (%)
Compound	1 h	2 h	3 h	4 h	Average potency in 4 h (%)
4g	96.6	131.0	131.4	139.1	124.5
4h	94.5	121.0	124.1	139.1	119.6
4i	70.3	105.2	94.6	94.8	91.2
5a	64.3	134.7	132.2	134.5	116.4
7	63.8	71.2	64.5	65.3	66.2
8	49.6	84.4	107.3	101.2	85.6
9	63.0	97.6	99.8	98.1	89.6
11	106.5	125.0	112.1	119.5	115.8
12	56.1	75.0	66.5	68.0	66.4
13	41.8	55.5	54.3	56.1	51.9
GTA	36.8	46.2	43.7	43.5	42.6
Indomethacin	100.0	100.0	100.0	100.0	100.0

Acknowledgments

We thank Prof. Laatsch, Institute of Organic and Biomolecular Chemistry, Goettingen for providing spectral and analytical data for the synthesized compounds. This work has been financed by the German Egyptian Scientific Projects (GESP), project No. 7. We are also thankful to Dr. Rehab Fawzy Abdel-Rahman, researcher in pharmacology department in NRC for her work in the pharmacological screening and statistical analysis.

References

Abubakirov N.K., Yatsyn V.K., . Uzb. Khim. Zh. 1959;5:80.
Adanin V.M., Khaletskii A.M.Zh., . Obshch. Khim.. 1967;5:1063.
Akamatsu H., Komura J., Asada Y., Niwa Y., . Planta Med.. 1991;57:119.
Aly A.M., Al-Alousi L., Salem H.A., . AAPS Pharm. Sci. Tech.. 2005;6:E74.
Baltina L.A., Flekhter O.B., Vasil’eva E.V., Tolstikov G.A., . Izv. Akad. Nauk SSSR, Ser. Khim.. 1997;9:2340.
Dalimov D.N., Dzhuraev A.Zh., Kamaev F.G., . Chem. Nat. Comp.. 2001;37:148.
Finney R.S., Tarnoky A.L.J., . Pharm. Pharmacol.. 1960;12:49.
Gheorghiu Th., Frotz H., Klein H.J., . Verh. Deut. Ges. Inn. Med.. 1971;77:511.
Ibrahim Y.A., Hanna A.G., Elgamal H.A., Allam R.F., Youssef Kh.A., . Pak. J. Sci. Ind. Res.. 1995;38:101.
Inoue H., Mori T., Shibata S.J., . Pharm. Pharmacol.. 1988;40:272.
Ishikawa S., Saito T., . Endocrinol. Jpn.. 1980;27:697.
Kase Y., Saitoh K., Ishige A., Komatsu Y., . Biol. Pharm. Bull.. 1998;21:1277.
Kondratenko R.M., Mustafina S.R., Baltina L.A., Vasil’eva N.G., Ismagilova A.F., Vasil’eva E.V., Nasyrov Kh.M., Galin F.Z., Tolstikov G.A., . Pharm. Chem. J.. 2001;35:243.
Maitraie D., Hung C., Yao Tu H., Liou Y., Wei B., Yang Sh., Wangd J., Nan Lin Ch.J., . Bioorg. Med. Chem.. 2009;17:2785.
Murav’ev I.A., Savchenko L.N., . Khim. Farm. Zh. 1979;5:78.
Ohuchi K., Tsurufuji A., . Mino Med. Rev.. 1982;27:188.
Ohuchi K., Kamada Y., Levine L., . Prostagland. Med.. 1981;7:457.
Okimasu E., Moromizato Y., Watanabe S., . Acta Med., Okayama. 1983;37:385.
Platonov V.G., Zorina A.D., Gordon M.A., . Khim-Farm. Zh.. 1995;29:42.
Rittenberg D., Ponticorvo I., . Proc. Natl. Acad. Sci. USA. 1960;46:822.
Schopke Th., Hiller K., . Pharmazie. 1990;45:313.
Shibata S., Takahashi K.S., Yano, Harada M., Saito H., Tamura Y., Kumagai A., Hirabayashi K., Yamamoto M., Nagata N., . Chem. Pharm. Bull.. 1987;35:1910.
Shon L.B., Kaplun A.P., Shpilevskii A.A., . Bioorg. Khim.. 1998;24:787.
Takahashi K., Shibata S., Yano S., Harada M., Saito H., Tamura Y., Kumagai A., . Chem. Pharm. Bull.. 1980;28:3699.
Tolstikov G.A., Baltina L.A., Shul’ts E.E., . Bioorg. Khim.. 1997;23:691.
Tolstikov G.A., Baltina L.A., Serdyuk N.G., . Khim-Farm. Zh.. 1998;32:5.
Tsukahara M., Nishino T., Furuhashi I., Inoue H., Sato T., Matsumoto H., . Chem. Pharm. Bull.. 2005;53:1103.
Ulmann A., Menard J., Corvol P., . Endocrinology. 1975;97:46-51.
Wang Z.Y., Nixon D.W., . Nutr. Cancer. 2001;39:1.
Winter C.A., Risley E.A., Nuss G.W., . Proc. Soc. Exp. Biol. Med.. 1962;111:544.
Yano Sh., Harada M., Watanabe K., . Chem. Pharm. Bull.. 1989;37:2500.

Introduction

Results and discussion

Chemistry
Biological activity

Materials and methods

Isolation of GTA from Glycyrrhiza glabra roots
Synthesis of the target derivatives
Anti-inflammatory testing

Fulltext Views
20

PDF downloads
13

View/Download PDF
Download Citations

BibTeX
RIS

Show Sections

[1] Abubakirov N.K., Yatsyn V.K., . Uzb. Khim. Zh. 1959;5:80.

[2] Adanin V.M., Khaletskii A.M.Zh., . Obshch. Khim.. 1967;5:1063.

[3] Akamatsu H., Komura J., Asada Y., Niwa Y., . Planta Med.. 1991;57:119.

[4] Aly A.M., Al-Alousi L., Salem H.A., . AAPS Pharm. Sci. Tech.. 2005;6:E74.

[5] Baltina L.A., Flekhter O.B., Vasil’eva E.V., Tolstikov G.A., . Izv. Akad. Nauk SSSR, Ser. Khim.. 1997;9:2340.

[6] Dalimov D.N., Dzhuraev A.Zh., Kamaev F.G., . Chem. Nat. Comp.. 2001;37:148.

[7] Finney R.S., Tarnoky A.L.J., . Pharm. Pharmacol.. 1960;12:49.

[8] Gheorghiu Th., Frotz H., Klein H.J., . Verh. Deut. Ges. Inn. Med.. 1971;77:511.

[9] Ibrahim Y.A., Hanna A.G., Elgamal H.A., Allam R.F., Youssef Kh.A., . Pak. J. Sci. Ind. Res.. 1995;38:101.

[10] Inoue H., Mori T., Shibata S.J., . Pharm. Pharmacol.. 1988;40:272.

[11] Ishikawa S., Saito T., . Endocrinol. Jpn.. 1980;27:697.

[12] Kase Y., Saitoh K., Ishige A., Komatsu Y., . Biol. Pharm. Bull.. 1998;21:1277.

[13] Kondratenko R.M., Mustafina S.R., Baltina L.A., Vasil’eva N.G., Ismagilova A.F., Vasil’eva E.V., Nasyrov Kh.M., Galin F.Z., Tolstikov G.A., . Pharm. Chem. J.. 2001;35:243.

[14] Maitraie D., Hung C., Yao Tu H., Liou Y., Wei B., Yang Sh., Wangd J., Nan Lin Ch.J., . Bioorg. Med. Chem.. 2009;17:2785.

[15] Murav’ev I.A., Savchenko L.N., . Khim. Farm. Zh. 1979;5:78.

[16] Ohuchi K., Tsurufuji A., . Mino Med. Rev.. 1982;27:188.

[17] Ohuchi K., Kamada Y., Levine L., . Prostagland. Med.. 1981;7:457.

[18] Okimasu E., Moromizato Y., Watanabe S., . Acta Med., Okayama. 1983;37:385.

[19] Platonov V.G., Zorina A.D., Gordon M.A., . Khim-Farm. Zh.. 1995;29:42.

[20] Rittenberg D., Ponticorvo I., . Proc. Natl. Acad. Sci. USA. 1960;46:822.

[21] Schopke Th., Hiller K., . Pharmazie. 1990;45:313.

[22] Shibata S., Takahashi K.S., Yano, Harada M., Saito H., Tamura Y., Kumagai A., Hirabayashi K., Yamamoto M., Nagata N., . Chem. Pharm. Bull.. 1987;35:1910.

[23] Shon L.B., Kaplun A.P., Shpilevskii A.A., . Bioorg. Khim.. 1998;24:787.

[24] Takahashi K., Shibata S., Yano S., Harada M., Saito H., Tamura Y., Kumagai A., . Chem. Pharm. Bull.. 1980;28:3699.

[25] Tolstikov G.A., Baltina L.A., Shul’ts E.E., . Bioorg. Khim.. 1997;23:691.

[26] Tolstikov G.A., Baltina L.A., Serdyuk N.G., . Khim-Farm. Zh.. 1998;32:5.

[27] Tsukahara M., Nishino T., Furuhashi I., Inoue H., Sato T., Matsumoto H., . Chem. Pharm. Bull.. 2005;53:1103.

[28] Ulmann A., Menard J., Corvol P., . Endocrinology. 1975;97:46-51.

[29] Wang Z.Y., Nixon D.W., . Nutr. Cancer. 2001;39:1.

[30] Winter C.A., Risley E.A., Nuss G.W., . Proc. Soc. Exp. Biol. Med.. 1962;111:544.

[31] Yano Sh., Harada M., Watanabe K., . Chem. Pharm. Bull.. 1989;37:2500.

Synthesis and biological activity of new 18β-glycyrrhetinic acid derivatives

Abstract

Keywords

18β-Glycyrrhetinic acid

Anti-inflammatory

1 Introduction

2 Results and discussion

2.1 Chemistry

2.2 Biological activity

3 Materials and methods

3.1 Isolation of GTA from Glycyrrhiza glabra roots

3.2 Synthesis of the target derivatives

3.2.1 Glycyrrhetinic acid acetate (2), acetyl glycyrrhetyl chloride (3) and methyl glycyrrhetate (10)

3.2.2 General procedures for the preparation of compounds 4a–j and 5a–e

3.2.3 N-(phenethyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4a)

3.2.4 N-(4-chlorophenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4b)

3.2.5 N-(benzyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4c)

3.2.6 N-(3-chloro-4-Methylphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4d)

3.2.7 N-(cyclohexyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4e)

3.2.8 N-(4-Methylphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4f)

3.2.9 N-(2-carboxyphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4g)

3.2.10 N-(5-Methyl isoxazol 3-yl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4h)

3.2.11 N-[2-(4-sulfamoylphenyl)ethyl]-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4i)

3.2.12 N-(morpholyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (4j)

3.2.13 (3,5-Dimethylphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5a)

3.2.14 (4-methylphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5b)

3.2.15 (4-ethyl carboxyphenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5c)

3.2.16 (4-chlorophenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5d)

3.2.17 (4-bromophenyl)-3β-acetyl-11-oxoolean-12-en-18β-H-30-oate (5e)

3.2.18 Preparation of N-(2-carboxyphenyl)-3β-hydroxy-11-ketoolean-12-en-18-βH-30-amide (6)

3.2.19 General procedures for the preparation of compounds 7 and 8

3.2.20 N-(2-carboxyphenyl)-3β-O-carboxypropanoyloxy-11-oxoolean-18β-H-30-amide (7)

3.2.21 N-(2-carboxyphenyl)-3β-O-phthaloyl-11-oxoolean-18β-H-30-amide (8)

3.2.22 Preparation of N-[2-(N-P-tolyl-benzamide)]-phenyl-3β-acetyl-11-oxoolean-12-en-18β-H-30-amide (9)

3.2.23 General procedures for the preparation of compounds 11–13

3.2.24 3-O-β-carboxypropionyl-11-oxoolean-12-en-18β-H-20β-O-methyl ester (11)

3.2.25 3-O-β-phthaloyl-11-oxoolean-12-en-18β-H-20β-O-methyl ester (12)

3.2.26 3-O-β-Carboxy-trans-propenoyl-11-oxoolean-12-en-18β-H-20β-O-methyl ester (13)

3.3 Anti-inflammatory testing

Acknowledgments

References

Suggested read for related articles: