Chemical modifications of natural triterpenes - glycyrrhetinic and boswellic acids: evaluation of their biological activity

Abstract

Synthetic analogues of naturally occurring triterpenoids; glycyrrhetinic acid, arjunolic acid and boswellic acids, by modification of A-ring with a cyano- and enone- functionalities, have been reported. A novel method of synthesis of α-cyanoenones from isoxazoles is reported. Bio-assays using primary mouse macrophages and tumor cell lines indicate potent anti-inflammatory and cytotoxic activities associated with cyanoenones of boswellic acid and glycyrrhetinic acid.

1. Introduction

Since the first report¹ of the synthesis of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) 1 and its methyl ester 2, there have been a number of studies displaying the multifarious biological activity of various analogues of this compound 1. Various derivatives of this compound possess significant biological activities. The CDDO-Im 3 triggers² apoptosis in multiple myelome (MM) cells. Compounds 1 and 3 are also highly active³ in suppressing cellular proliferation of human leukemia and breast cancer cell lines. CDDO 1 and its methyl ester 2 exhibit^4,5,6 high inhibitory activity against production of nitric oxide induced by interferon-γ (IFN-γ) in mouse macrophages (IC₅₀ = 0.1 nM). CDDO 1 was also found^7,8 to induce monocytic differentiation of human myeloid leukemia cells and adipogenic differentiation of mouse 3T3-L1 fibroblasts. The compound CDDO 1 was synthesized from an abundantly occurring triterpene, oleanolic acid. SAR studies⁶ conducted on CDDO have mainly addressed to the structural variations at C-2 of the A ring, the functionality in C-ring and the ester moiety. We now report the synthesis of the compounds, 5 from glycyrrhetinic acid 8, 6 from arjunolic acid 16, and 7 from boswellic acids 26 (Scheme 1), which are readily available from Indian medicinal plants. These cyano-enones 5, 6 and 7 have been screened for nitric oxide inhibitory and cytotoxic activities in in vitro bioassays.

Scheme 1.

Cyano-enone derivatives of triterpenes

2. Results and Discussion

With a view to study the effect of changes in the position of the carboxylic moiety and increase in the polarity of the A ring, three naturally abundant triterpenoids from Indian medicinal plants were chosen as substrates for the synthesis of the CDDO analogues. Since glycyrrhetinic acid 8, isolated from liquorice root,⁹ already has the α,β-unsaturated system in the C ring, it was only necessary to introduce a cyano-enone functionality in the A ring of the triterpenoid. The formal synthesis of the cyano-ketone was carried out using the Johnson’s¹⁰ protocol via the isoxazole intermediate as depicted in Scheme 2. Glycyrrhetinic acid 8, derived from the acidic hydrolysis of glycyrrhizin, was esterified using diazomethane in quantitative yields. The ester 9 thus obtained was oxidized at 0 °C using the Jones Reagent to give the 3-keto derivative 11 in 84% yield. The compound was conversely prepared by reversing the oxidation and esterification procedures to initially give the keto acid 10 that was esterified to give the keto ester 11. The formylation of the keto ester using ethyl formate in the presence of an alkoxide base gave 2-formyl-3-keto compound 12, the precursor for the isoxazole in 80% yield. The isoxazole 13 was prepared¹¹ in an acetate buffer as reported by Doorenbos in 82% yield to give the single desired isomer. This was quantitatively isomerized under basic conditions to the cyanoketone 14 that was dehydrogenated using DDQ in benzene to give the cyano enone ester 5 in 65% yield. An alternative protocol for the synthesis of the cyanoenone 5 was attempted via the allylic bromination-dehydrobromination. The isoxazole 13 was brominated using NBS under radical conditions and the crude product 15 gave the cyanoenone 5 when treated with a base in comparable yields.

Scheme 2.

Reagents: a) i) CH₂N₂/MeOH; ii) Jones oxidation b) NaOMe/HCOOEt; c) NH₂OH/AcOH; AcONa/MeOH/C₆H₆; d) NaOMe/MeOH/Et₂O; e) DDQ/C₆H₆; f) NBS/AIBN/CHCl₃

Spectroscopic data established the assigned structure which was further supported by x-ray crystal structure analysis (Figure 1). This also revealed that the stereochemistry¹² of the starting material 8 had been retained in the product without any epimerization of the CD rings taking place under the basic conditions of the reactions.

Figure 1.

Crystal structure of methyl 2-cyano-3,11-dioxo-30-norolean-1,12-dien-30-oate 5

Arjunolic acid 16, isolated¹³ from Terminalia arjuna, while retaining an olean skeleton with a C-28 carboxylic acid has an additional hydroxyl group (C-23) in the A ring providing additional polar functionality. It was initially attempted to selectively activate the C-2 hydroxy and displace with a nitrile, however various attempts gave intractable mixtures. As an alternate protocol, the hydroxyl group at C-2 of methyl arjunolate 17, was removed following the Barton’s xanthate protocol. Initially, the two hydroxyl groups at C-3 and C-23 of 17 were protected as an acetonide 18 which readily formed a xanthate ester 19 with carbon disulfide. Reduction of the xanthate ester 19 with tributyltin hydride, followed by hydrolysis afforded the diol 21 in good yield. This compound was identical with methyl hederagenin, obtained by the esterification of hederagenin, a natural triterpene isolated¹⁴ from Sapindus emarginatus. Selective protection of the primary alcohol as its TBDMS ether 22, followed by the oxidation of the secondary alcohol at C-3 using PDC afforded the ketone 23. Following the Johnson’s protocol¹⁰, the isoxazole 25 was prepared which subsequently afforded the desired cyano-enone 6 in good yield as depicted in Scheme 3. The silyl protecting group was cleaved under the acidic conditions during the preparation of the isoxazole.

Scheme 3.

Reagents: a) CH₂N₂/Et₂O; b) acetone, HCl; c) CS₂/NaH, imidazole;d) TBTH, AIBN, PhH; e) Amberlite resin; f) t-BDMSi-Cl, imidazole, CH₂Cl₂; g) PCC/CH₂Cl₂; h) HCOOEt, NaOtAm; i) NH₂OH.HCl/MeOH; j) (i) NaOMe; (ii) DDQ/C₆H₆.

Boswellic acids, isolated¹⁵ from the gum of Boswellia serrata, were obtained as a mixture of α and β isomers in the ratio of (9:1), belonging to the olean and ursane skeletons and differing only in the position of the methyl groups in the ring-E of the triterpene moiety. Both α and β isomers however retain a carboxylic moiety as a pendant at C-4 in the A ring of the triterpenoid. The synthesis of the cyano-enone derivative of boswellic acid was depicted in Scheme 4. The free acids 26 were esterified to furnish 27 in order to ensure safe oxidation of the C-3 hydroxyl group to the keto group 28. Formylation of 28 afforded the compound 29. Since this compound decomposed on standing, the crude compound was directly employed in the subsequent steps without purification. Thus, reaction of 29 with hydroxylamine hydrochloride in refluxing methanol gave the isoxazole 30. Bromination-dehydrobromination protocol was employed to afford the desired mixture of cyanoenones 7 of boswellic acids in a (9:1) ratio as indicated from its nmr spectrum. Further work on the purification of 7 is in progress.

Scheme 4.

Reagents: a) CH₂N₂/MeOH; b) PDC/CH₂Cl₂; c) HCOOEt/NaOMe; d) NH₂OH/HOAc e) NBS/AIBN/CHCl₃; f) NaOMe/CH₃OH

3. Bioassays

In a previous study Suh et al.⁷ reported that 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) shows potent anti-inflammatory, anti-proliferative, apoptotic and differentiating activities. Subsequently its methyl and imidazolide analogues have been found to be more potent in these activities than CDDO.^3,8,16,17 Here we have screened 5, 6 and 7 for their ability to inhibit nitric oxide production (Table 1) induced by IFN-γ in primary mouse macrophages and for cytotoxicity using MTT assay (Table 2).

Table 1.

Inhibition of IFN-γ induced Nitric Oxide production by modified triterpenoids

Triterpenoids	EC50
Boswellic acids (26a and 26b)	No effect till 20 μM
Methyl 3-oxoboswellate (28)	No effect till 20 μM
Isoxazole from methyl boswellate (30)	No effect till 20 μM
Cyano-enone from methyl boswellate (7)	0.074 μM
Glycyrrhetinic acid (8)	>20 μM
Isoxazole from methyl glycyrrhetinate (13)	>20 μM
Cyano-ketone from methyl glycyrrhetinate (14)	14.5 μM
Cyano-enone from methyl glycyrrhetinate (5)	0.173 μM
Arjunolic acid (16)	>20 μM
Cyano-ketone of methyl arjunolate	7.5 μM
Isoxazole of methyl arjunolate (25)	8.7 μM
Cyano-enone from methyl arjunolate (6)	1.6 μM

Table 2.

Cytotoxic actitivities of 5, 6 and 7 (EC₅₀ values) on different tumor cell lines as determined by MTT assay.

No.	Cell line	Description	EC50 (nM) of the compounds
			7	5	6
1	A549	Lung carcinoma	599	7598	7936
2	A431	Skin carcinoma	505	4271	1697
3	HL60	Leukemia	108	925	548
4	MCF-7	Breast carcinoma	522	6213	2249
5	T47D	Breast carcinoma	327	5541	3907
6	HT1080	Acetabulum fibro sarcoma	393	7747	3029

As shown in table 1, 7 shows highest activity (EC₅₀ 74.14 nM) for the inhibition of Nitric Oxide followed by 5 (EC₅₀=172. 8 nM). In contrast 6, showed lower potency (EC₅₀ 1.6 μM). All others (intermediates) showed activities at > 5 μM. The ability of the modified triterpinenes to inhibit growth has been demonstrated previously. Hence, we tested the growth inhibitory/cytotoxic activities of modified triterpenoids derived from boswellic acid (7), glycyrrhetinic acid (5) and arjunolic acid (6) using MTT assay. As shown in table 2, compound 7 shows a potent activity with an EC₅₀ of 0.108 μM on HL60 leukemia cells. The cytotoxic activity of 5 and 6 on HL60 was much less at EC₅₀ of 0.925μM and 0.548 μM respectively. In other cell lines the EC₅₀ ranged from 0.3 to 0.6μM for 7 and 4.2 to 7.8 μM for 5 and 1.7–8.0 μM for 6 (Table-2). These results suggest that 7 has a very potent anti inflammatory and growth inhibitory activities among the three modified triterpenoids. Although, cyano-enone modification results in more potent activities of the triterpenoids, not all the compounds are comparable. This is obvious when we compare CDDO⁷ and 5–7 of this study. Recently, modified betulinic acid was reported¹⁸ to have a very potent activity as compared to CDDO. Taken together, modified triterpenoids are a good source of lead molecules for drug development targeting inflammation and cancer.

Experimental

General methods: Melting points were recorded on a Buchi B-540 apparatus and are uncorrected. Infrared spectra were recorded on JASCO FTIR 410. Proton (¹H NMR) and carbon magnetic resonance (¹³C NMR) spectra were generally recorded on a JEOL JNM-LA 300 spectrometer. Mass spectra measurements were carried out on a JEOL JMS DX 303 spectrometer. Elemental analyses were carried out on a Carlo Erba Element Analyzer 1106 at the department of organic chemistry, Indian Institute of Science. Analytical thin-layer chromatography (TLC) was performed on (10 x 5 cm) glass plates coated with Acme silica gel G or GF254 (containing 13% calcium sulfate as binder). Visualization of the spots on TLC plates was achieved either by exposure to iodine vapor or UV light or by spraying sulfuric acid and heating the plates at 120°C. Column chromatography was performed by using Acme silica gel (100–200 mesh) or neutral alumina. All solvents were freshly distilled over CaH₂ or Na/benzophenone as appropriate. X-ray data were collected at 293K on a SMART CCD–BRUKER diffractometer with graphite monochromated MoKα radiation (γ =0.7107Å). Structure was solved by direct methods (SIR92). Refinement was by full-matrix least-squares procedures on F² using SHELXL-97. The non-hydrogen atoms were refined anisotropically whereas hydrogen atoms were refined isotropically.

Glycyrrhetinic acid, (3β-hydroxy-11-oxo-30-norolean-12-en-30-oic acid) 8⁹

Mp. 295°C; ν_max(neat)/cm⁻¹ 3440, 2945, 1705, 1664, 1457, 1386 and 867; ¹H NMR (300 MHz; CDCl₃): δ 5.72 (1H, s), 3.21 (1H, dd, J 9.0 and 6.8 Hz), 2.80 (1H, d, J 13.5 Hz), 2.32 (1H, s), 2.21–0.66 (42H, m); ¹³C NMR (75 MHz; CDCl₃): δ 199.6, 181.0, 168.5, 128.4, 100.4, 85.7, 78.6, 61.7, 55.0, 48.0, 45.3, 43.6, 43.0, 40.9, 39.0, 37.6, 37.0, 32.7, 31.8, 30.8, 28.5, 28.3, 28.0, 27.3, 26.5, 23.4, 18.6, 17.4, 16.2 and 15.5; m/z (DI) 470 (M⁺), 303, 262, 175 and 135 (100).

3,11-Dioxo-30-norolean-12-en-30-oic acid 10

Jones reagent (prepared from 18.24 g of CrO₃) was added drop wise to a mechanically stirred suspension of glycyrrhetinic acid 7 (9.21 g, 19.6 mmol) in acetone (400 mL) at 0°C over a period of 30 min till the brown color persisted. The mixture was stirred for further 30 min. Propan-2-ol (5 mL) was added, reaction mixture filtered, and the residue was thoroughly washed with dichloromethane. The solution was concentrated to 100 mL and was diluted with dichloromethane (400 mL). The organic layer was washed with water (2 x 200 mL), brine and dried over sodium sulfate. The organic layer was concentrated under vacuum and the residual solid was recrystallized from methanol/dichloromethane to give the pure ketone 10 (8.43 g, 92%) as a colorless crystalline solid. mp 308–310 °C (from dichloromethane/methanol); (Found: C, 76.7; H, 9.5. C₃₀H₄₄O₄ requires C, 76.9; H, 9.4%); ν_max(KBr)/cm⁻¹ 3311, 2965, 1726, 1682, 1644, 1455 and 1386; ¹H NMR (300 MHz; CDCl₃): δ 5.75 (1H, s), 2.97 (1H, m), 2.64 (1H, m), 2.41 (1H, s), 2.41–0.87 (40H, m); ¹³C NMR (75 MHz; CDCl₃): δ 217.3, 199.7, 181.2, 169.8, 128.4, 76.6, 61.0, 55.4, 48.2, 47.8, 45.3, 43.8, 43.3, 40.9, 39.7, 37.7, 36.7, 34.2, 32.1, 31.9, 30.9, 28.6, 28.4, 26.5, 26.3, 23.3, 21.4, 18.8, 18.5 and 15.6.

Methyl 3β-hydroxy-11-oxo-30-norolean-12-en-30-oate 9

A solution of ethereal diazomethane (in excess) was added to a suspension of glycyrrhetinic acid 7 (9.4 g, 20 mmol) in methanol (150 mL) at 0°C. The reaction mixture was allowed to stand overnight. The excess of diazomethane was quenched with acetic acid (6 drops) and the mixture was concentrated under vacuum. The solid was recrystallized from methanol/dichloromethane mixture to give colorless crystals 9 (9.6 g, 99%). mp 254–256°C (from dichloromethane/methanol); (Found: C, 76.65; H, 10.1. C₃₁H₄₈O₄ requires C, 76.85; H, 9.9%); ν_max(neat)/cm⁻¹ 3357, 2946, 1722, 1656 and 1467; ¹H NMR (300 MHz; CDCl₃): δ 5.63 (1H, s), 3.69 (3H, s), 3.20 (1H, m), 2.80 (1H, d, J 13.5 Hz), 2.30 (1H, s) and 2.15–0.65 (41H, m); ¹³C NMR (75 MHz; CDCl₃): δ 199.2, 176.2, 168.2, 128.5, 78.5, 61.7, 54.9, 51.5, 48.2, 45.2, 43.9, 43.0, 41.1, 39.1 (2 C), 37.7, 37.0, 32.8, 31.7, 31.0, 28.5, 28.2, 28.1, 27.3, 26.4, 23.4, 18.6, 17.4, 16.3 and 15.6; m/z (DI) 484 (M⁺), 317, 276, 175 and 135 (100%).

Methyl 3,11-dioxo-30-norolean-12-en-30-oate 11

Following the procedure for the synthesis of 10 from 8 described earlier, the oxidation of the methyl ester 9 (9.21 g, 19 mmol) gave 11 as amorphous solid (8.80 g, 95%) [This compound was found to be identical to that obtained by diazomethane esterification of the acid 10]. mp 248–250 ºC (from dichloromethane/methanol); (Found: C, 77.6; H, 9.8. C₃₁H₄₆O₄ requires C, 77.2; H, 9.5%); ν_max(neat)/cm⁻¹ 2942, 1725, 1704, 1655, 1625, 1463 and 1386; ¹H NMR (300 MHz; CDCl₃): δ 5.68 (1H, s), 3.69 (3H, s), 2.97 (1H, m), 2.62 (1H, m), 2.40 (1H, s) and 2.37–0.83 (39H, m); ¹³C NMR (75 MHz; CDCl₃): δ 215.6, 198.4, 176.1, 168.7, 128.3, 60.9, 55.3, 51.5, 48.2, 47.5, 45.1, 43.8, 43.2, 41.1, 39.6, 37.7, 36.6, 33.9, 32.1, 31.7, 31.0, 28.5, 28.2, 26.5, 26.4, 26.3, 23.3, 21.3, 18.7, 18.5 and 15.5; m/z (DI) 482 (M⁺), 317, 276 and 135(100%).

Methyl 2-hydroxymethylene-3,11-dioxo-30-norolean-12-en-30-oate 12

To an ice-cold suspension of sodium methoxide (440 mg, 8 mmol) in dry benzene (20 mL) was added a solution of ketone 11 (1.9 g, 4 mmol) and ethyl formate (634 mg, 8 m mol) over a period of 10 min under an atmosphere of nitrogen. The resulting mixture was allowed to stand overnight at room temperature. Ice water (20 mL) was added and the two layers separated. The organic layer was extracted with 20% sodium hydroxide solution (3 × 20 mL). The combined aqueous layer was acidified with 2N hydrochloric acid and extracted with ethyl acetate (3 x 20 mL). The ethyl acetate layer was washed with water, brine, dried over sodium sulfate and concentrated under vacuum. Recyrstallization of the solid from dichloromethane/methanol afforded the pure colourless formyl derivative 12 (1.60 g, 78%). mp 232–234 °C (from dichloromethane/methanol); (Found: C, 75.1; H, 9.15. C₃₂H₄₆O₄ requires C, 75.3; H, 9.0%); ν_max(KBr)/cm⁻¹ 3438, 2934, 1732, 1653, 1618, 1457 and 1386; ¹H NMR (300 MHz; CDCl₃): δ 14.84 (1H, d, J 3.0 Hz), 8.62 (1H, d, J 3.0 Hz), 5.71 (1H, s), 3.71 (3H, s), 3.46 (1H, d, J 14.1 Hz), 2.40 (1H, s) and 2.15–0.84 (38H, m); ¹³C NMR (75 MHz, CDCl₃): δ 198.5, 189.2, 188.2, 176.1, 169.1, 128.4, 105.5, 59.5, 52.2, 51.5, 48.1, 44.8, 43.8, 43.1, 41.2, 39.9, 39.6, 37.7, 36.1, 31.7, 31.6, 31.0, 28.5, 28.4, 28.1, 26.4, 26.3, 23.2, 20.8, 18.7, 18.2 and 14.6; m/z (DI) 510 (M⁺), 317, 276, 135 and 83 (100%).

Isoxazole derivative of the methyl ester of glycyrrhetinic acid 13

The formyl derivative 12 (0.80 g, 1.6 mmol) in benzene (6 mL) was mixed with a solution of hydroxylamine hydrochloride (134 mg, 1.92 mmol), sodium acetate (312 mg, 1.92 mmol) in acetic acid (1 mL) and water (1 mL). The mixture was homogenized by the addition of methanol (2 mL) and refluxed for a period of 5h. The solvents were removed under reduced pressure and the residue was diluted with water (15 mL) and extracted with ethyl acetate (3 x 15 mL). The combined organic layer was washed with brine, dried over sodium sulfate and filtered. The solvent was removed under vacuum and the resulting solid was recrystallized from dichloromethane/methanol to give a colourless solid 13 (0.64 g, 80%). mp 279–280°C (from dichloromethane/methanol); (Found: C, 75.7; H, 9.05; N, 2.4. C₃₂H₄₅NO₄ requires C, 75.7; H, 8.9; N, 2.7%); ν_max(neat)/cm⁻¹ 2972, 1730, 1658, 1620, 1460, 1385 and 870; ¹H NMR (300 MHz; CDCl₃): δ 7.97 (1H, s), 5.72 (1H, s), 3.71 (3H, s), 3.65 (1H, d, J 15.1 Hz), 2.50 (1H, s) and 2.16–0.84 (38H, m); ¹³C NMR (75 MHz; CDCl₃): δ 198.4, 176.2, 171.7, 169.0, 149.8, 128.5, 108.8, 59.9, 53.2, 51.5, 48.1, 45.1, 43.8, 43.2, 41.2, 38.3, 37.7, 35.9, 34.6, 31.8 (2 C), 31.0, 28.8, 28.5, 28.2, 26.5, 26.4, 23.2, 21.5, 18.2, 18.0 and 15.6; m/z (DI) 507(M⁺), 135 and 83 (100%).

Methyl 2-cyano-3,11-dioxo-30-norolean-1,12-dien-30-oate 5

The isoxazole 13 (270 mg, 0.5 mmol) in diethyl ether (5 mL) was added to a stirred solution of sodium (60 mg, 2.6 mmol) in dry methanol (3 mL) under an atmosphere of argon at 0°C. It was allowed to stir for 1 h. Cold water (5 mL) was added to the reaction mixture and the organic layer was washed thoroughly with 5% potassium hydroxide solution (3 x 10 mL). The combined alkaline extracts were acidified and extracted with ethyl acetate (3 x 15 mL). The organic layer was washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The crude residue 14 (248 mg, 91%) was directly used in the next step without purification. ν_max (KBr)/cm⁻¹ 2963, 2204, 1726, 1657, 1461, 1388 and 801; ¹H NMR (300 MHz; CDCl₃): δ 5.70, (1H, s), 3.70 (3H, s), 2.40 (1H, s) and 2.34–0.83 (40H, m); m/z (DI) 507(M⁺), 310, 276, 135 (100). A mixture of the crude cyano-ketone 14 (63 mg, 0.12 mmol) and DDQ (33 mg, 0.14 mmol) in benzene (3 mL) was refluxed for 8 h. After cooling the reaction mixture to ambient temperature, acetone was added till it became homogenous and the clear solution was filtered through a pad of alumina. The filtrate was concentrated and the solid residue was recrystallized from dichloromethane/methanol to give the pure cyano-enone 5 (40 mg, 65%). mp 237–239 °C (from dichloromethane/methanol); (Found: C, 76.1; H, 8.5; N, 2.8, C₃₂H₄₃NO₄ requires C, 76.0; H, 8.6; N, 2.8%); ν_max(neat)/cm⁻¹ 2951, 2232, 1727, 1685, 1655, 1610, 1457, 1387, 802 and 736; ¹H NMR (300 MHz; CDCl₃): δ 8.49 (1H, s), 5.78 (1H, s), 3.71 (3H, s), 2.70 (1H, s) and 2.20–0.84 (37H, m); ¹³C NMR (75 MHz; CDCl₃): δ 197.4, 196.8, 176.2, 171.9, 170.9, 127.8, 114.5, 113.3, 54.2, 51.7, 51.6, 48.3, 45.5, 44.9, 43.8, 43.4, 41.1, 39.5, 37.6, 31.7, 31.6, 31.0, 28.5, 28.1, 27.4, 26.4, 26.2, 23.3, 21.4, 19.4, 18.8 and 18.0; m/z (DI) 83 (100%).

Crystal Data

Compound 5: C₃₂H₄₃O₄N, MW = 505, colorless crystal, Crystal system: monoclinic, space group: P2(1), cell parameters: a = 7.282 (4) Å, b = 12.262 (8) Å, c = 16.173 (10) Å, β = 94.57° (1), V = 1440.00 (6) ų, Z = 2, D_c = 1.162 g.cm⁻³, F(000)=544.0, μ=0.08 mm⁻¹. Total number of l.s. Parameters = 342, R1 = 0.0494 for 4676 Fo > 4σ (Fo) and 0.0655 for all 5812 data. WR2=0.1230, GOF = 1.019, Restrained GOF = 1.024 for all data. Crystallographic data (without structure factor) have deposited with the Cambridge Crystallographic Data Center and the depository number is CCDC 213948.

Synthesis of 5 via allylic bromination–dehydrobromination

A mixture of the isoxazole 13 (270 mg, 0.5 mmol), NBS (98 mg, 0.55 mmol), AIBN (catalytic amount) in chloroform (5 mL) was allowed to reflux under the influence of tungsten lamp for 6 h. The solution was cooled, and the precipitated succinimide was filtered Removal of the solvent afforded the crude bromocompound 15 which was added to an ice-cold solution of sodium (46 mg, 2 mmol) in methanol (2 mL). The reaction mixture was stirred at room temperature for one hour. The solvent was removed under vacuum and the residue was diluted with water (10 mL). The aqueous layer was repeatedly extracted with ether (3 x 15 mL). The combined organic layer was dried over sodium sulfate and concentrated under vacuum. The crude solid was purified by column chromatography to give the cyano-enone 5 (63%). Mp. 237–239°C.

Arjunolic acid 16¹³

Arjunolic acid was isolated¹³ from the acetone extract of the heartwood of Terminalia arjuna Bedd. It was crystallized from acetone, mp 320–325 °C; ν_max(KBr)/cm⁻¹ 3544, 3466, 3389, 2931, 1708, and 1639; ¹H NMR (300 MHz; py-d5): δ 5.48 (1H, br t), 4.25–4.23 (3H, s), 3.74 (1H, d, J 10.2 Hz), 3.30 (1H, d, J 10.2 Hz), 2.34 (1H, d, J 9.6 Hz), 2.18–0.92 (41H, m); m/z (DI) 248 (M⁺), 203 (100), 191, 173, 133.

Methyl arjunolate 17

Ethereal diazomethane was added to an ice cooled suspension of arjunolic acid 16 (9.8 g, 2 mmol) in methanol (25 mL). The resultant mixture was left at room temperature for 4 h. The excess diazomethane was quenched with drops of acetic acid. Removal of the solvent afforded a residue which was crystallized from methanol to give the methyl ester 17 (9.5 g, 95%).¹³ mp 248–250 °C; ν_max(neat)/cm⁻¹ 3395, 2946 and 1725; ¹H NMR (300 MHz; CDCl₃):δ 5.28 (1H, br s), 3.71 (1H, br s), 3.61 (3H, s), 3.57 (1H, br s), 3.48 (2H, m), 2.84 (1H, d, J 13.2 Hz), 1.95–0.67 (41H, m); ¹³C NMR (75 MHz; CDCl₃): δ 178.3, 144.0, 122.3, 80.1, 69.8, 69.7, 58.3, 51.7, 48.1, 47.7, 46.9, 46.0, 41.9, 41.5, 39.5, 38.3, 38.2, 34.0, 33.1, 32.7, 32.5, 30.7, 28.2, 27.8, 26.1, 23.6, 23.5, 23.2, 18.4, 17.1 and 12.9.

Preparation of the isopropylidene derivative 18

A mixture of methyl arjunolate 17 (500 mg, 1 mmol) in dry acetone (50 mL) containing a catalytic amount of conc. hydrochloric acid and activated 4Å molecular sieves was stirred for 6 h. The reaction mixture was filtered, concentrated and the residue was purified by column chromatography (hexane/ethyl acetate, 7/3) to give 18 (412 mg, 76%); mp 179–181°C (from ethyl acetate); (Found: C, 75.3; H, 10.1; C₃₄H₅₄O₅ requires C, 75.2 and H, 10.0%); Rf (30% ethyl acetate/hexane 0.4); ν_max(neat)/cm⁻¹ 3492, 2945 and 1725; ¹H NMR (300 MHz; CDCl₃): δ 5.28 (1H, br t), 3.78 (1H, dt, J 4.5 and 9.7 Hz), 3.62 (3H, s), 3.61 (1H, d, J 9.7 Hz), 3.48 (1H, d, J 3.3 Hz), 3.32 (1H, d, J 9.3 Hz), 2.87 (1H, dd, J 14.4 and 5.5 Hz), 2.23 (1H, s), 2.07–0.88 (44H, m); ¹³C NMR (75 MHz; CDCl₃): δ 177.6, 143.4, 122.0, 99.4, 82.0, 72.5, 64.9, 51.4, 51.3, 47.6, 46.4, 46.2, 45.7, 41.5, 41.1, 39.3, 37.9, 36.8, 33.8, 33.1, 32.2, 32.0, 30.6, 29.6, 27.5, 25.9, 23.6, 23.1, 22.9, 19.3, 17.7, 17.4, 16.7 and 13.4.

Xanthate derivative 19

A solution of 18 (1.1 g, 2 mmol), sodium hydride (240 mg, 10 mmol), imidazole (50 mg, 0.74 mmol) in THF (10 mL) was stirred for 30 min. and freshly distilled carbon disulphide (1 mL, excessive amount) was added and the mixture was heated under reflux for 2 h. The reaction mixture was cooled to ambient conditions and methyl iodide (1.5 mL, excess) added. The mixture was further heated under reflux for 1 h. Water (15 mL) was added to the cooled mixture and extracted with ether (3 x 20 mL). The organic layer was washed with brine and dried. Removal of the solvent under reduced pressure afforded a residue which was purified by chromatography on silica gel (hexane/ethyl acetate, 10/1) to obtain 19 (1.16 g, 92%). mp 214–216°C; (Found: C, 68.55; H, 9.0; C₃₆H₅₆O₅S₂ requires C, 68.3 and H, 8.9%); ν_max(neat)/cm⁻¹ 2946, 1725 and 1197; ¹H NMR (300 MHz; CDCl₃):δ 5.78 (1H, dd, J 10.3 and 4.2 Hz), 5.26 (1H, br s), 3.80 (1H, d, J 10.3 Hz), 3.62 (3H, s), 3.44 (1H, d, J 10.3 Hz), 2.86 (1H, dd, J 4.0 and 13.0 Hz), 2.51 (3H, s), 2.36 (1H, dd, J 4.8 and 12.3 Hz), 2.00–0.70 (44H, m); ¹³C NMR (75 MHz; CDCl₃):δ 178.2, 143.7, 121.8, 99.4, 78.8, 78.6, 77.20, 72.6, 51.5, 51.1, 47.7, 46.6, 45.8, 43.4, 41.6, 41.2, 39.4, 38.8, 37.8, 33.8, 33.1, 32.3, 32.0, 30.7, 29.7, 27.6, 26.9, 26.0, 23.6, 23.3, 23.0, 19.2, 18.5, 17.4, 17.3, 16.6 and 13.5.

Methyl hederagenin 21 (from arjunolic acid)

A catalytic amount of AIBN was added to a mixture of 19 (1.0 g, 1.58 mmol) and TBTH (0.5 mL) in dry benzene (10 mL). The reaction mixture was heated under reflux for 2 h, the solvent was removed under reduced pressure and the residue refined by chromatography (hexane/ethyl acetate, 10/1) to obtain a white powder 20 (673 mg, 81%). This compound was sufficiently pure to be used for the subsequent operation. ν_max(neat)/cm⁻¹ 2942 and 1724; ¹H NMR (300 MHz; CDCl₃): δ 5.28 (1H, t, J 3.6 Hz), 3.62 (3H, s), 3.47 (1H, m), 3.53 (1H, d, J 10.0 Hz), 3.44 (1H, d, J 10.0 Hz), 2.86 (1H, dd, J 14.0 and 4.8 Hz), 2.04–0.70 (46H, m). Amberlite-450 was added to 20 (600 mg, 1.14 mmol) in methanol (6 mL) and the resultant mixture allowed to stand at ambient temperature for 12 h with occasional shaking. The mixture was filtered through a pad of celite and the filtrate concentrated under reduced pressure. The resultant residue was purified by column chromatography (hexane/ethyl acetate, 3:2) to give methyl hederagenin as a colorless solid 21 (482 mg, 87%).¹⁴ mp 240–242°C; Rf (40% ethyl acetate/hexane 0.42); identical with an authentic sample prepared from hederagenin, ν_max(neat)/cm⁻¹ 3507, 2942 and 1724; ¹H NMR (300 MHz; CDCl₃): δ 5.28 (1H, br t), 3.74 (1H, d, J 10.0 Hz), 3.63 (1H, br s), 3.62 (3H, s), 3.44 (1H, d, J 10.0 Hz), 2.86 (1H, dd, J 13.2 and 3.3 Hz), 2.32–0.70 (42H, m); ¹³C NMR (75 MHz; CDCl₃): δ 178.5, 144.2, 122.4, 118.2, 80.2, 69.6, 69.5, 51.5, 48.0, 47.6, 46.5, 45.0, 41.7, 41.5, 39.6, 38.3, 38.2, 34.0, 33.4, 32.5, 32.3, 30.3, 28.4, 27.8, 26.2, 23.5, 23.4, 23.1, 17.1 and 13.1.

Isolation of hederagenin (3,23-dihydroxyolean-12-en-28-oic acid)

Dry pericap of soapnuts, Sapindus mukorossi, (S. emarginatus) abundantly available in southern states of India were collected. The pulverized, dried pericaps of soap nuts (125 g) were defatted by extraction with hexane (200 mL) in a soxhlet extractor. This was further extracted with ethyl acetate (300 mL) followed by methanol (300 mL). The methanol extract (16.2 g) was submitted for hydrolysis with 6N hydrochloric acid (60 mL) for 2 h. The acidic mass was carefully neutralized with 5% sodium hydroxide solution and extracted with ethyl acetate (5 x 60 mL). The combined ethyl acetate extract was evaporated and dried under vacuum. The crude sapogenin, hederagenin (6.5 g) was crystallized from methanol to afford pure hederagenin, mp 333–335 °C; ν_max(neat)/cm⁻¹ 3447, 2930, 1698 and 756; ¹H NMR (300 MHz; CDCl₃): δ 5.46 (1H, br s), 4.22–4.24 (3H, m), 3.72 (1H, d, J 10.2 Hz), 3.32 (1H, d, J 10.2 Hz), 2.34 (1H, d, J 9.6 Hz), 2.16–0.91 (41H, m); m/z (DI) 248, 203 (100%), 191, 173, 133.

Methyl hederagenin 21 (from hederagenin)

Ethereal diazomethane was added to an ice-cold suspension of hederagenin (600 mg, 1.20 mmol) in methanol (4 mL). The resultant mixture was left at ambient temperature for 4 h. The excess diazomethane was quenched using drops of acetic acid. The solvent was removed by evaporation under reduced pressure and the residue purified by column chromatography on silica gel (hexane/ethyl acetate, 3:2) to give the methyl ester (570 mg, 95%). This compound was identical with compound 21.¹⁴ mp 240–242°C.

23-O-t-butyldimethylsilyl derivative of methyl hederagenin 22

tert-Butyldimethylsilyl chloride (159 mg, 1.06 mmol) in dichloromethane (1 mL) was added to a mixture of the diol 21 (432 mg, 0.88 mmol) and imidazole (108 mg, 1.59 mmol) in dry dichloromethane (1.5 mL) at 0°C over a period of 30 min. The reaction mixture was stirred for 6 h and worked up to give a residue which was purified by chromatography. Elution with hexane/ethyl acetate (9/1) afforded a viscous oil 22 (480 mg, 91%), (Found: C, 73.94; H, 10.73; C₃₇H₆₄O₄Si, requires C, 74.0 and H, 10.33%); Rf (5% ethyl acetate/hexane 0.3); ν_max(neat)/cm⁻¹ 3420, 2949 and 1726; ¹H NMR (300 MHz; CDCl₃): δ 5.27 (1H, br t), 3.67 (1H, d, J 9.0 Hz), 3.61 (3H, s), 3.60 (1H, d, J 5.4 Hz), 3.35 (1H, d, J 9.0 Hz), 2.85 (1H, dd, J 14.0 and 4.0 Hz), 2.16 (1H, s), 1.95–0.70 (55H, m).

Methyl 23-O-t-butyldimethylsilyl-3-oxoolean-12-en-28-oate 23

A solution of alcohol 22 (376 mg, 0.67 mmol) in dry dichloromethane was added to mixture of pyridinium chlorochromate (731 mg, 3.40 mmol) and sodium acetate (730 mg, 8.9 mmol) in dry dichloromethane. The reaction mixture was stirred for 4 h before filtering through a pad of celite. The concentrated residue was refined by column chromatography on silica gel (hexane/ethyl acetate, 95/5) to give viscous oil 23 (292 mg, 78%). (Found: C, 73.65; H, 10.75; C₃₇H₆₂O₄Si requires C, 74.25 and H, 10.36%); Rf (5% ethyl acetate/hexane 0.5); ν_max(neat)/cm⁻¹ 2927, 2856, 1731 and 1703; ¹H NMR (300 MHz; CDCl₃) 5.33 (1H, br t), 3.62 (3H, s), 3.61 (1H, d, J 9.0 Hz), 3.27 (1H, d, J 9.0 Hz), 2.86 (1H, m), 2.38 (2H, m), 2.04–0.8 (53H, m).

Isoxazole of methyl hederagenin 25

A mixture of the ketone 23 (250 mg, 0.42 mmol), ethyl formate (0.2 mL, excess) and sodium tertiary amylate {prepared from sodium (48 mg, 2.09 mmol) and t-amyl alcohol (1.5 mL)}in benzene (2 mL) was stirred at room temperature for 8 h. The reaction mixture was acidified with 2N hydrochloric acid and extracted with ether (3 x 15 mL), washed with brine and dried over anhydrous sodium sulfate. The organic layer was concentrated to afford a glassy solid 24 which was directly used in the next step. ν_max(neat)/cm⁻¹ 2948, 2856 and 1728; ¹H NMR (300 MHz; CDCl₃): δ 8.31 (1H, d, J 3.3 Hz), 5.30 (1H, br s), 3.70 (1H, d, J 9.0 Hz), 3.63 (3H, s), 3.62 (1H, br s), 3.34 (1H, d, J 9.0 Hz), 2.95-2.85 (1H, m), 2.21–0.80 (55H, m). The crude formyl derivative 24 in methanol (3 mL), hydroxylamine hydrochloride (58 mg, 0.84 mmol) and sodium acetate (68 mg, 0.84 mmol) were heated under refluxed for 8 h. The reaction mixture was cooled and the solvent removed under reduced pressure. The residue in water (6 mL) was extracted with ethyl acetate (15 x 3 mL). The combined organic layers were washed with water, brine and dried over anhydrous sodium sulfate. The residue was purified by column chromatography. Elution with (hexane/ethyl acetate, 4/1) afforded the pure isoxazole 25 (146 mg, 68% for two steps). mp 218–220°C; (Found: C, 74.95; H. 9.3; N, 2.45; C₃₂H₄₇NO₄ requires C, 75.4; H. 9.3 and N. 2.75%); Rf (20% ethyl acetate/hexane 0.5); ν-_max(neat)/cm⁻¹ 2974, 1732, 1656, 1620, 1460, 1385 and 870; ¹H NMR (300 MHz; CDCl₃): δ 8.02 (1H, s), 5.34 (1H, br t), 3.80 (1H, d, J 11.0 Hz), 3.63 (3H, s), 3.55 (1H, d, J 11.0 Hz), 2.9 (1H, dd, J 4.0 and 14.4 Hz), 2.5–0.8 (39H, m); m/z(DI) 507 (M⁺), 310, 276, 135 (100).

Methyl 2-cyano-23-hydroxy-3-oxooleana-1,12-dien-28-oate 6

The cyano-ketone (179 mg, 96%), Rf (20% ethyl acetate in hexane 0.3) was prepared from the isoxazole 25 (186 mg, 0.36 mmol) by hydrolysis using sodium methoxide in methanol. Since this compound could not be purified, it was directly used in the next step. The crude cyano-ketone (155 mg, 0.30 mmol) was oxidized using DDQ to give the cyano-enone 6 (98 mg, 65%). mp 221–223 °C; (Found: C, 75.5; H, 8.7; N, 2.6; C₃₂H₄₅NO₄ requires C, 75.7; H, 8.9 and N, 2.8%); Rf (20% ethyl acetate in hexane 0.25) ν_max(neat)/cm⁻¹ 2951, 2232, 1727, 1685, 1655, 1610 and 802; ¹H NMR (300 MHz; CDCl₃): δ 7.74 (1H, s), 5.34 (1H, t, J 3.3 Hz), 3.76 (1H, d, J 10.8 Hz), 3.63 (3H, s), 3.62 (1H, br s), 3.43 (1H, d, J 10.8 Hz), 2.89 (1H, dd, J 5.7 and 14.7 Hz), 2.15–2.12 (2H, m), 2.00–1.92 (2H, m), 1.71–0.83 (32H, m); ¹³C NMR (75 MHz; CDCl₃): δ 198.0, 177.6, 169.5, 144.8, 121.0, 115.0, 114.3, 67.4, 51.6, 50.2, 46.7, 45.7, 45.3, 42.2, 41.6, 41.3, 40.5, 40.4, 34.0, 33.4, 32.3, 32.0, 30.9, 27.8, 26.0, 23.8, 23.4, 23.0, 19.1, 18.5, 17.6 and 17.2.

Isolation of β-boswellic acid from the gum of Boswellia serrata:¹⁵

The commercially available gum (100 g) of Boswellia serrata was extracted with ethyl acetate (3 x 100 mL) and the ethyl acetate extract was filtered and concentrated under vacuum. The syrupy mass (45 g) was treated with 5% sodium hydroxide solution and stirred till a uniform emulsion is formed. The emulsion was extracted with hexane (3 x 100 mL). The aqueous extract was acidified with dilute hydrochloric acid and the precipitate was extracted with ethyl acetate (3 x 100 mL). The ethyl acetate extract was thoroughly washed with water, dried over anhydrous sodium sulfate and concentrated to afford a creamy powder (22 g) consisting a mixture of boswellic acids. This mixture (5 g) was chromatographed on a silica gel column. Elution with hexane-ethyl acetate (4:1) afforded a mixture of β-, and α-boswellic acids (2.2 g) in the ratio of 9:1, Rf (20% ethyl acetate/hexane 0.3).

Methyl boswellates 27

The methyl esters of α- and β-boswellic acids 27 (606 mg, 98%) were obtained from boswellic acids 26 (602 mg, 1.32 mmol) by esterification with ethereal diazomethane.¹⁵ m.p 195°C; (Found: C, 78.83; H, 10.50; C₃₁H₅₀O₃ requires C, 79.10; H, 10.78%); Rf (20% ethyl acetate/hexane 0.6); ν_max(neat)/cm⁻¹ 3511, 2921 and 1722; ¹H NMR (300 MHz; CDCl₃): δ 5.13 (1H, s), 4.10 (1H, br s), 3.61 (3H, s), 2.23–0.78 (45H, m); m/z (DI) 470 (M⁺), 394, 161.

Methyl 3-ketoboswellates 28

A solution of the alcohol 27 (658 mg, 1.40 mmol) and PDC (602 mg, 1.60 mmol) were taken in dry dichloromethane (6 mL). The reaction mixture was stirred for 4 h before filtering through a pad of celite. The concentrated residue was purified by column chromatography on silica gel (20% ethyl acetate in hexane) to give a colorless solid 28 (467 mg, 71%).¹⁵ m.p. 160°C; (Found: C, 79.35; H, 10.25; C₃₁H₄₈O₃ requires C, 79.40; H, 10.30%); Rf (20% ethyl acetate/hexane 0.8); ν_max(neat)/cm⁻¹ 2949, 1742, 1714 and 1662; ¹H NMR (300 MHz; CDCl₃): δ 5.54 and 5.30 (1H, br s), 3.68 (3H, s), 2.92 (1H, dt, J 6.0 and 14.7 Hz), 2.34 (1H, d, J 14.7 Hz Hz), 2.16–0.76 (42H, m); ¹³C NMR (75 MHz; CDCl₃): δ 207.9, 173.9, 139.9, 124.0, 59.2, 58.2, 57.4, 52.0, 46.6, 42.3, 41.5, 41.1, 39.9, 39.7, 37.2, 36.5, 33.9, 33.0, 31.3, 28.9, 28.1, 26.6, 23.7, 23.2, 21.5, 21.0, 20.6, 17.6, 16.9 and 13.4; m/z (DI) 468 (M⁺), 218 (100%).

Preparation of the isoxazole 30

Employing a protocol similar to the synthesis of 25 from 23, the crude of the 2-hydroxy-methylene derivative 29 was obtained from 28 (456 mg, 0.98 mmol) which was directly used for the synthesis of isoxazole 30 (358 mg, 74%). (Found: C, 77.8; H, 9.6; N, 2.85; C₃₂H₄₇NO₃ requires C, 77.9; H, 9.53; N, 2.84%); Rf (20% ethyl acetate/hexane 0.57); ν_max(neat)/cm⁻¹ 2918, 1732 and 1646; ¹H NMR (300 MHz; CDCl₃): δ 8.05 (1H, s), 5.24 and 5.19 (1H, br t), 3.63 (3H, s), 2.53 (1H, m), 2.15–0.77 (41H, m); ¹³C NMR (75 MHz; CDCl₃): δ 172.9, 166.9, 150.1, 139.7, 123.9, 121.27, 111.2, 77.2, 59.1, 55.3, 52.1, 47.3, 46.8, 46.3, 45.1, 42.2, 41.8, 41.4, 39.8, 39.7, 39.5, 38.2, 38.2, 37.0, 35.9, 35.6, 34.6, 33.8, 33.3, 32.5, 32.4, 32.0, 31.2, 31.1, 28.8, 28.4, 28.1, 26.9, 26.6, 26.1, 25.6, 24.4, 23.6, 23.0, 21.3, 19.8, 17.4, 16.4, 16.3, 14.5 and 14.3; m/z (DI) 493 (M⁺), 218 (100%).

Cyano-enone of methyl boswellates 7

Following the bromination-dehydrobromination protocol employed for the synthesis of 4 from 13, the cyano-enone of α- and β-methyl boswellates 7 (90 mg, 54% yield) was obtained from 30 (174 mg, 0.34 mmol) as an amorphous solid. The isomers were observed in a (1:9) ratio, based on the integration of the ¹H proton signals. (Found: C, 78.4; H, 9.0; N, 2.5. C₃₂H₄₅NO₃ requires C, 78.2; H, 9.2; N, 2.85%); Rf (20% ethyl acetate/hexane 0.4); ν_max(neat)/cm⁻¹ 2922, 2234, 1729, 1698 and 1612; ¹H NMR (300 MHz; CDCl₃): δ 7.62 and 7.60 (1H, s), 5.17 and 5.12 (1H, br t), 3.59 (3H, s), 2.17–0.79 (40H, m); ¹³C NMR (75 MHz; CDCl₃): δ 190.3, 172.5, 168.0, 146.0, 140.7, 122.5, 120.0, 114.7, 113.6, 59.0, 54.5, 54.4, 54.3, 52.5, 47.3, 46.5, 42.5, 41.4, 41.3, 41.0, 40.8, 40.7, 39.54, 39.5, 36.9, 34.6, 33.7, 33.3, 32.6, 32.5, 32.3, 31.6, 31.1, 31.0, 28.3, 27.8, 26.9, 26.6, 26.4, 25.9, 25.7, 23.6, 23.5, 23.3, 23.1, 22.6, 21.3, 20.9, 19.3, 17.5, 17.4, 14.1, 14.0 and 13.9; m/z (DI) 491 (M⁺), 476, 432, 218 (100%), 203.

BIOASSAYS

Nitric Oxide assay

Thioglycolate (4%) elicited peritoneal macrophages were isolated from two months old C57black6 male mice after sacrificing by cervical dislocation. The cells were cultured for 72h in presence of mouse interferon gamma (10U/ml) either alone or in presence of test compounds. Induced nitric oxide rapidly oxidized into nitrite in culture medium (RPMI+10%FBS) and the nitrite concentration in supernatants was taken as a measure of nitric oxide production. Griess assay¹⁰ was done to measure the nitrite in the medium.

MTT assay for cytotoxicity

Cancer cell lines were plated in 96-well plates (Nunc, Denmark) at a density of 5 x 10³ – 8 x 10³ cells/well depending on the cell line. The plates were incubated at 37°C in a humidified 5% CO₂ atmosphere. Cells were allowed to adhere to the surface for 3–4 h and different concentrations of the compounds were added and incubated for 72 h. Later, 20μL of MTT solution (5mg/mL) was added to each well and the incubation continued for an additional 3h. The dark blue formazan crystals formed within the healthy cells were solubilized with DMSO and the absorbance was measured in an ELISA plate reader at 550 nm. The EC50 values of the compounds were calculated by Graphpad Prism.