International Journal of Drug Development and Research

Keywords

Cedrelone; Semi-synthetic; Antibacterial; Antifungal.

Introduction

The plant Toona ciliata Roemer belongs to the family Meliaceae is a tall tree grown in hotter parts of India. It is used for various medicinal purposes like emmenagogue, menstrual disorder and in various forms of ulceration, anti periodic in fever, rheumatism, tonic, beneficial in chronic dysentery, blood complaints (Ayurveda), cardiotonic, aphrodisiac, anthelmentic; good for scabies and expectorant (Yunani) [1]. The chemical composition of T.ciliata are known [2-7]. In the present study a series of cedrelone derivatives were synthesized from cedrelone which was isolated from Toona ciliata heartwood. And the compound was characterized by IR, 1H NMR, 13C NMR and Mass spectra. The compounds were screened for antibacterial and antifungal activities by disc diffusion method. The aim was find out the better antimicrobial agent from the naturally occurring compound cedrelone.

Chemistry

The compound cedrelone was isolated from T.ciliata hexane extract and the different derivatives prepared from cedrelone were (1) 1, 2-dihydrocedrelone, (2) cedrelone ethyl ether, (3) bromohydroxy cedrelone, (4) cedrelone Michael adduct, (5) 5, 6-dihydro cedrelone and (6) cedrelone 1, 2-epoxy acetate. The synthetically modified products were characterized by spectroscopic techniques like 1H NMR, 13C NMR, IR and Mass spectral data.

Antimicrobial activity

Antimicrobial test was carried out by disc diffusion method [8] using the inoculums containing seeded nutrient agar medium. The antibacterial activity was tested against Gram positive a) Staphylococcus aureus (ATCC 9144), b) Staphylococcus epidermitis (ATCC 155), c) Bacillus subtilis (ATCC 6633), d) Bacillus cereus (ATCC 11778), e) Micrococcus luteus and Gram negative organisms a) Escherichia coli (ATCC 25922), b) Pseudomonas aeruginosa (ATCC 9027). Fungi organism a) Candida albicans (ATCC 1091), b) Aspergillus niger (ATCC 6275). The antimicrobial activities were measured by the zone of inhibition expressed in millimeter around the disc. All experiments were carried out in duplicates.

Results and Discussion

The compound cedrelone was isolated from the hexane extract of powdred heartwood of Toona ciliata; the extract was concentrated under vacuum and column chromatograhed over silica gel to give pure cedrelone, m.p.213ºC; Crude product yield 0.4%, mp 213ºC. Cedrelone was characterized by the following spectral data. IR (KBr) cm-1: 3396 cm-1 (hydroxyl) 1675 cm-1 (a,b unsaturated carbonyl) 1621 cm-1, 1606 cm-1 (-C=C-), 2982 (methyl), 872 cm-1, 1463 cm-1, 3150 cm-1 (furan moiety) 1365 cm-1, 1382 cm-1 (C-O stretching). 1H NMR (200 MHz, CDCl3)δ: 6.92 (d,1H;J=10Hz ,C-1), 6.12 (d,1H;J=10Hz,C-2), 2.94 (m,1H;C-9), 2.20 (m,2H;C-11), 2.15-2.66 (m,2H;C-12), 4.0(s,1H;C-15), 1.50-1.90(m,2H;C-16), 1.81(m,1H;C-17), 7.45 (s,1H;C-21), 6.54 (d,1H;C-22), 7.33 (s,1H;C-23), 1.53 (s,3H;CH3), 1.43 (s,3H;CH3), 1.29 (s,3H;CH3), 1.1 (s,3H;CH3), 0.9 (s,3H;CH3). 13C NMR (200MHz,CDCl3) δ: 152.3 (C-1), 127.2 (C-2), 197.9 (C-3), 48.7 (C-4), 133.8 (C-5), 141.1 (C-6), 203.6 (C-7), 46.7 (C-8), 41.7 (C-9), 41.9 (C-10), 20.1 (C-11), 35.1 (C-12), 40.2 (C-13), 69.7 (C-14,C-O), 55.0 (C-15), 35.1 (C-16), 43.1 (C-17), 123.1 (C-20), 139.3 (C-21), 110.6 (C-22), 142.9 (C-23), 26.7, 23.8, 23.0, 21.1, 19.4 (5CH3). The scheme of semisynthetic modification was given in Fig.1.

1, 2-dihydrocedrelone (Compound-1)

Reduction of the carbon- carbon double bond in conjugated systems by sodium borohydride has been previously reported [9]. When we attempted to reduce cedrelone-using excess of sodium borohydride at room temperature only 1, 2 dihydrocedrelone was obtained. A systematic analysis of spectral data of the product indicated that the 13C NMR values, the appearance of carbon signals at 22.9 and 31.8 while signals due to α, β- unsaturated double bond (152.3 and 127.2) was missing. The 1H NMR spectral data of the compound 1 with that of starting material showed clearly the absence of peak at 6.2 and 6.8 due to α, β- unsaturated double bond and presence of peak at 2.05 and 2.92 for 1,2 dihydrocedrelone. The compound shows that the absence of IR band at 1675 cm-1 (a,b unsaturated double bond), and the M+ 424 of the compound confirm the structure.

Cedrelone ethyl ether (Compound-2)

The enolic group present in B ring of cedrelone reacted with diethyl sulphate in alkaline medium to give the compound cedrelone ethyl ether. The compound was crystallized after passing through column packed with neutral alumina. A comparison of both 1H and 13C NMR spectra of compound 2 with that of cedrelone, revealed only the differences in the resonance values due to B ring of cedrelone. The extra resonance at 66.7 ppm (OCH2) and methyl resonance at 18.8 ppm indicated the formation of cedrelone ethyl ether. The 1H NMR spectral data of the ether compound appearance of signal at 3.86- 4.30 ppm (OCH2CH3) and 1.29 ppm (OCH2CH3) was observed. The IR indicates the disappearance of peak due to OH group at 3440 cm-1 and the presence of IR band at 1687 cm-1 for The M+ 450 of the compound confirms the structure. 4.3. Bromohydroxy cedrelone (Compound-3) An attempt was made to apply the polymer supported bromide resin (I) for the desired bromohydroxylation reaction.

Ketones treated with resin I or II in methanol, at either room temperature or under reflux conditions, yielded both the bromomethoxylated product and bromohydroxy compound [10]. However when the resins IRA 400 (Cl) Resin BTP (India) Ltd, Chennai - 600 017 was used for bromination of cedrelone in acetonitrile: water (1:1) system at room temperature only bromohydroxylated compound was obtained in moderate yield. The 13C NMR values of the product indicated that the appearance of carbon signals at 76.4 and 61.1 while signals due to α, β-unsaturated double bond (152.3 and 127.2) were missing. The 1H NMR spectral data of the compound 3 with that of starting material showed clearly the absence of peak at 6.2 and 6.8 due to α, β-unsaturated double bond and presence of peak at 4.14 and 4.55 for bromohydroxylated cedrelone. The IR absorptions showed the absence of peak at 1694 cm-1 and presence of peak at 720 cm-1, 1763 cm-1 (-C-C-Br) and M+ 519 of the compound confirms the structure.

Cedrelone Michael adduct (Compound - 4)

Ethyl-2-N-(2-hydroxybenzyl) amino-3-methyl butanoate synthesized from our laboratory was used for various syntheses. However, this reaction was extended to cyclic α, β-unsaturated ketone, unsaturated double bond and the presence of IR band at 1720 cm-1 (COOEt), 1681 cm-1 (-C=O), 1615 cm-1 and the M+ 582 of the compound confirms the structure. The 13C NMR values of the product indicated that the appearance of carbon signals at 41.6 and 31.8 while signals due to α, β-unsaturated double bond at 152 and 127 were missing. Moreover the additional peak at 168.2, 129.6, 62.1 and 22.2 [- CH (COO CH2CH3)2], confirms the possible addition product. The 1H NMR spectral data of the compound 4 with that of starting material showed clearly the absence of peak at 6.92 and 6.12 due to α, β- unsaturated double bond and presence of peak at 2.14 and 2.53 for cedrelone Michael adduct. The compound showed the absence of IR band at 1675 cm-1 due to α, β- unsaturated double bond and the presence of IR band at 1720 cm-1 (COOEt), 1681 cm-1 (-C=O) and the M+ 582 of the compound confirms the structure.

5, 6-dihydrocedrelone (Compound -5)

In the present study we attempted to use the inert N, N-dimethylaniline borohydride (amineborane) for reaction with cedrelone under microwave irradiation condition and obtained selective reduction at C5-C6 double bond. Interestingly the other groups such as 1,2 -double bond in A ring, ketene and epoxide functional group remained insensitive. The 13C NMR values of the product indicated the appearance of carbon signals at 71.2 and 81.5 while signals due to α, β-unsaturated double bond (133.8 and 141.1) were missing. The 1H NMR spectral data of the compound 5 with that of starting material showed clearly the presence of peak at 2.19 and 4.34 for 5, 6- dihydrocedrelone. The M+ 424 of the compound confirms the structure.

Cedrelone 1, 2-epoxy acetate (Compound -6)

Cedrelone1, 2-epoxide on acetylation with acetic anhydride and pyridine the corresponding acetate was obtained at room temperature. The 13C NMR of both the substrate and the product exhibited the same number of carbon signals to that of cedrelone1, 2-epoxide, except for the changes in the carbon and proton signals due to acetate. It indicates that enolic group present in B ring of 1, 2-epoxy cedrelone has modified due to acetylation. The extra carbonyl resonance at 168.4 ppm and methyl resonance at 25.3 ppm indicated the formation of acetate. A comparison of 1H NMR of compound 6 with that of starting material showed clearly the presence of peak at 2.26 indicates the product formation, while the rest of the molecule remained intact. Further the IR shows the absence of OH group (3440, 1749cm-1). The compound was well agreed with the MS (M+ 498).

Antimicrobial Activity

Antimicrobial Activity of semi-synthetically modified compounds was given in Table 1. Cedrelone and its derivative exerted inhibitory response against all the species of organism tested, except E. coli. It is the least susceptible organism as compared to other organism. The degree of antifungal activity of bromohydroxy cedrelone and Michael adduct were pronounced and the activity was greater than that observed with other compounds tested.

Experimental protocols

Chemistry

Melting points were determined on a Toshniwal melting point apparatus (Toshniwal Pvt.Ltd, India) and were uncorrected. NMR spectra were recorded on a Bruker 200 MHz instrument using TMS as the internal standard. CDCl3 was used as the solvent. Chemicals shifts were given in terms of parts per million (δ scale). Mass spectrum were recorded on a Shimadzu QP 1000A mass spectrometer. IR spectra were recorded using IFS 85 Bruker FTIR instrument. Solid samples were recorded as KBr pellets. Column chromatography was performed using silica gel (60- 120 mesh, 70-325 mesh, 230-400 mesh, 400-600 mesh). Precoated silica gel plates (E-Merck, Germany, Art 5554 Kieselgel 60F254, 0.2 mm thickness) were used for thin layer chromatography. UV irradation (λ 254 nm and 366 nm), 10% H2SO4 in alcohol, ammonium hepta molybdate – cerric suphate in H2SO4 were used. A modified microwave oven with a magnetic stirrer was used for carrying out reactions.

Isolation

Dried and powdered heartwood of Cedrela toona was extracted with hexane, the hexane extract was concentrated under vacuum. Crude extract was about 0.4% of the weight of dried heartwood. The extract was column chromatographed over silica gel to get pure cedrelone. Yield: 0.3%. mp 213ºC. The pure cedrelone was used for the synthesis of the following compounds.

Method of synthesis

1, 2-dihydro cedrelone (TBAB reduction) (Compound -1)

Cedrelone (100 mg) and TBAB (170 mg) were dissolved in dry THF and the reaction was allowed to takes place at room temperature for 2 h. The reaction was monitored by TLC. Addition of water precipitated crystalline product dihydrocedrelone, identical with the compound obtained by sodiumborohydride reduction. (Yield 70 mg), mp 202ºC; IR (KBr) cm-1: 3379 (hydroxyl), 2937 (methyl), 1699 1615 (-C = C-), 872, 1453 (furan moiety) 1345 (-C-O stretching. MS m/z (M+): 424 (Calcd for C26 H32O6: 424.5). Anal. C26H30O6.

Cedrelone ethyl ether (Compound -2)

Cedrelone (125 mg, 0.3 mmole) and diethyl sulphate (0.25 ml) were dissolved in dioxane (3 ml). Small addition of (0.3 ml) of 1 N potassium hydroxide solution was made at intervals sufficient to maintain yellow colour of the solution. When addition of alkali no longer re-established the colour further quantity of 1ml was added to hydrolyse the excess of diethyl sulphate. Dissolution of the resulting precipitate in benzene –chloroform (1:1) followed by filteration through neutral alumina and evaporation, gave cedrelone ethyl ether (80 mg), which was crystallized from chloroform-ethanol gave cedrelone ethyl ether. (Yield : 60 mg). mp225-226ºC; IR (KBr) Cm-1: 2934(methyl), 1687 1584(C=C), 873, 1445(furan moiety), 1307, 1381(-C-O stretching). MS m/z (M+): 451 (Calcd for C28 H34O5: 450.6). Anal. C28 H34 O5.

Bromohydroxy cedrelone (Compound - 3)

Cedrelone (150 mg, 0.36 mmole), polymer supported bromine chloride (P-Br2Cl- 750 mg) and 5 ml of acetonitrile: water (1:1) system was stirred at room temperature. The reaction was monitored by TLC solvent system hexane: ethyl acetate (7:3). Acetonitrile was removed by rotovac. The aqueous portion was partitioned with chloroform. The chloroform layer on evaporation gave bromohydroxy cedrelone. It was crystallized with chloroformethanol gave a reddish brown crystalline compound. (Yield 35 mg). IR (KBr) Cm-1: 3391(hydroxyl), 2922(methyl), 1740(-C=O), 1618(C=C), 789, 1459(furan moiety), 1354 (-C-O stretching), 720 (-CC- Br). MS m/z (M+): 519 (Calcd for C28 H31O6 Br: 519.4). Anal. C28 H31O6Br

Cedrelone Michael adduct (Compound - 4)

Ethyl-2-N- (2-hydroxybenzyl) amino 3-methyl butanoate synthesized in our laboratory (237 mg) in dry THF was added to a solution of LiAlH4 (38 mg) in dry THF. The mixture was stirred at room temperature for 1h, followed by addition of cedrelone (212 mg) and diethyl malonate (212 mg) and stirring was continued for another 4 h. Reaction was performed in an oven dried side arm flask, closed with a septum and fitted with a take off adapter. Reaction was carried out in nitrogen atmosphere. The reaction was quenched with 1 N HCl and the mixture was extracted with ethyl acetate [11]. The organic layer was washed successively with saturated NaHCO3 solution, brine and dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure gave a syrupy compound, which on crystallization gave a pure solid. (Yield 50 mg). IR (KBr) Cm-1: 3369 cm-1 (hydroxyl) 2931 cm-1 (methyl), 1720 cm-1 (O=C-OEt), 1681 cm-1 (-C=O), 1615 cm-1 (C=C), 917, 1445 cm-1 (furan moiety), 1355 cm-1, 1386 cm-1 (C-O stretching). MS m/z (M+): 582 (Calcd for C33 H42O9: 582.6). Anal. C33 H42 O9.

Amine boration (Compound -5)

Cedrelone (108 mg) and N, N-dimethyl aniline borohydride (0.25 m mole) was taken in a 100 ml conical flask fitted with a condenser and kept in a microwave oven [11]. The reaction was monitored every 2 minutes by TLC, using hexane-ethylacetate (7:3) as solvent system. The reaction was completed in 8 minutes. Then the reaction mixture was quenched with methanol. Removal of methanol yielded a solid, which was washed with hexane followed by 2% ethyl acetate to remove the unused reagent. The solid obtained after washing was analysed for its structure. The synthesized compound was confirmed by spectral data. (Yield: 40 mg). IR (KBr) Cm-1: 3369 cm-1 (hydroxyl) 2931 cm-1 (methyl), 1720 cm-1 (O=C-OEt), 1681 cm-1 (-C=O), 1615 cm-1 (C=C), 917, 1445 cm-1 (furan moiety), 1355 cm-1, 1386 cm-1 (C-O stretching) 1615 (-C=C-). MS m/z (M+): 424 (Calcd for C26 H30O5: 424.5). Anal. C26 H30O5.

Cedrelone 1, 2-epoxy acetate (Compound -6)

Cedrelone 1, 2 epoxide synthesized from our laboratory was used for the synthesis of cedrelone 1, 2 epoxy acetate. 1, 2-epoxy cedrelone 105 mg (0.24 m mole) was taken and acetylated with acetic anhydride (0.2-0.3 ml) and pyridine (1 ml), stirred at room temperature for 30 min. The solution was poured into water and extracted with ether. The extract was washed with dilute sulphuric acid, sodium bicarbonate solution and water. Drying and removal of solvent and crystallization of the crude product with ethanol gave crystalline compound cedrelone 1, 2-epoxy acetate (Yeild-80%). mp 190°C; IR (KBr) Cm-1: 2983 (methyl), 1756 (enolate acetate), 1680 (a,b-unsaturated carbonyl), 1615 (-C=C-), 1716 (epoxy ketonyl), 875, 1445 (furan moiety), 1355, 1386 (C-O stretching). MS m/z (M+): 480 (Calcd for C28 H32O7: 480.5). Anal. C28 H32 O7.

Screening of antimicrobial activity of the semi synthetic compound

The antimicrobial activity of the synthesized compounds was tested against S. aureus, S. epidermitis, B. subtilis , B. cereus, M. luteus and Gram -ve organisms E. coli, P. aeruginosa using nutrient agar medium (M 087 Hi-Media laboratories, India) final pH at 25 ?C is 7.3 ± 0.2.The antifungal activity of the compounds was tested against C. albicans and A. niger using Sabouraud dextrose-agar medium (M063) (Himedia), final pH at 25 ?C is 5.6 ± 0.2

Antimicrobial test was carried out by the disc diffusion method using the inoculums containing seeded nutrient agar medium [8]. The dried plant extracts were dissolved in dimethyl formamide (DMF), (50 mg/ml) 5 μl (250 μg/disc) was applied to the sterile filter paper disc (diameter, 6 mm). The discs were placed on the inoculated nutrient agar / Sabouraud dextrose medium along with solvent control and an appropriate reference antibiotic disc was applied for bacteria and fungi. For bacteria ciprofloxacin 10 μg /disc and for fungi ketoconazole 10 μg /disc served as a positive control. The filter paper disc impregnated with DMF was used as solvent control. The bacterial petri dishes were incubated at 37.5 ºC for 24 h and fungi petri dishes were incubated at 37 ºC for 24-48 h. The antimicrobial activities were measured by the zone of inhibition expressed in millimeter around the disc. All experiments were carried out in duplicates.

Tables at a glance

Table 1

Figures at a glance

Figure 1

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