Indian Journal of Chemistry Vol. 38B, February 1999, pp. 160 - 165

Microbiological degradation of a synthetic benzofurane type lactone

S Solujic*, S Sukdolak & L J Krstict

Department of Chemistry, University of Kragujevac, P.O. Box 60, YU-34000 Kragujevac, Yugoslavia

tlnstitute of Chemistry, Technology and Metallurgy Nagoseva 12 YU-llOOO, Belgrade, Yugoslavia

Received 12 February 1997; accepted 5 January 1998

A benzofurane type lactone 4 incorporating an u-methylene-y-butyrolactone moiety has been synthesized. The key steps involved are the Michael condellsation of 4-dichloromethyl-4-methyl-2,5-cyclohexadienone 1 with dimethyl malonate and acetoxylation of 4-dichloromethyl-5-carboxymethyl-4-methyl-2-cyclohexenone 2 with lead (IV) acetate in BFr etherate which are indicative of the general route to y-Iactone. Microbial degradations of the synthetic benzofurane type lactone 4 have also been studied using Aspergillus niger. Saccharomyces cerevisiae. Bacillus mycoides. Agrobaclerium tumefaciens, Pseudomonas glicinea and Pseudomonas flu orescens. TIle lactone acts both as an additive for the nutrient substrate and as a source of organic carbon.

Physiological actiVity of the natural lactones of plant origin is known eversince santonin was used as an important antihelminatic and ascaricidol agent. Because of their high level of physiological activity,

natural and synthetic lactones have been the ~ubject of biochemical investigations as potential therapeutic

agents since 19701-3

From the chemistry point of view, potential

cytotoxic activity of many sesquiterpene lactones of plant origin is due to their ability to perform selective inhibition of certain enzyme systems . Many results published so far prove that the factor directly responsible for the cytotoxic activity of such compounds is the presence of O=C-C=CH2 system in the molecule4-6.

The physiological actIvIty of lactones increases with the presence of more than one group of unsaturated ester or epoxide in the five-membered or six-membered rings of the molecule . It has also been demonstrated that synthetic a-methylenebutyro-lac tone derivatives containing no other reactive functional group can have growth inhibition activity comparable to that of multifunctional products 7-10.

The results published in this field, to date, have opened the way to the formulation of new synthetic

methods for obtaining lactone, a-methylenebutyro-lactone and similar functional compounds which

. provide some therapeutic effects as inhibitors of cellsin culture of CCRF-CEM human lymphoblastic leukemia"

12.

Our investigation on bi o logical act I vltles of lactones started with microorganisms because of

several favourable aspects : short vegetation pe ri od , ability of enzyme system to adapt to a ltered development conditions, selective me thods for investigation of biochemical parameters and suffic ient biological materia l for investigation .

The aim of the present work was to investi gate the biochemical reactions of the microorganisms deve-ioped in nutrient substrate inter alia containing lactone 4 as well as in substrates in which 4 was used as the source of organic carbon .

The investigation on extracellular samples pointed out to the ability of the tested fungi and bacteria to biodegrade the synthetic lactone 4 , and based on the chemical structures of degradation products the bi odegradation mechanisms of lactone were proposed I } .

Chemical assay

In our laboratory , we achieved a chemical synthesis of the benzofurane type lactone with a new structure blonging to the biologically active lac tones of the

SOLUJIC et al. : MICROBIAL DEGRADA nON OF A SYNTHETIC BENZOFURANE TYPE LACTONE 161

type a-methylenebutyrolactone. We have also reported the chemical transformation of 4-dichloromethyl- 4 -methyl- 5 -( dimethoxycarbonyl )-methylene-2-cyclohexenone 1 in the synthesis of several polyfunctional products with y-Iactone ring.

The most direct route to y-Iactone 4 seems to be selenium dioxide oxidation of 4-dichloromethyl-4-methyl- 5 -methylenecarboxy- 2 -cyclohexenone 2 (Scheme I) in view of the work of Bhalerao and Rapopart on the allylic oxidation of trisubstituate 0Iefins'4. A modest yield of compound 3, (3H, 3aH, 7a H- 4 -methyl- 4 -dichloromethyl- 2, 7 -benzofuran-dione) was obtained from the oxidation of 2 with I equiv . of selenium dioxide in refluxin g dioxane. Oxidative acetoxylation '5 of 2 with lead (TV) acetate in abso lute benzene in the presence of a catalytic amount of BF, etherate at 20C during 5 hr . gave the

y-Iactone 3 as a white crystalline product; only trallS-3H,3aH,7aH- 4 -dic hloromethyl-4-methyl-2,7-benzo-furandione 3 in 75-80% yie ld .

Both ox idati on and cyc li zation reactions took place simultaneously; the 6-trans acetoxy lated product 4-dichloromethyl-4-methyl-5-methyl enecarboxy-6-trans-acetoxy-2-cyc lohexenone was not isolated .

The y-Iactone derivative 2~ was formylated ' 6. ' 7 by standard technique to give the sodium salt of a-

formyl--y-Iacton e (non-isolated) in 65.5 % yie ld . Subsequent re fluxin g with formaldehyde gave the desired lactone 4 as white crystals in 56% yie ld .

The reaction of a-formy llactone sod ium sa lt with formaldehyde for producing the exocyclic methylene double bond, may be best ex plained by formyl group transfer from carbon to oxygen followed by elimination of carboxy late an ion ' H

iv - o 7. , 6' 3

(i) C H2 (COOCH .. )2. CH .. OH/Na; (ii ) HCI; (iii) Pb(OAc k BFr El2 0; (iv) a : HCOOEI. Na H; B: HC HO.THF

Scheme I

o

The resultant compounds were characterized by their physical and spectral (I3C, 'H NMR and [R) data.

Earlier, the stereochemistry of substituents at C-4 and C-5 in compound 2 was established as lrans-equatorial 19.20. The structure of the lactone 3 was established on the basis of the following data. The 'H

NMR spectrum exhibited a singlet at 0 1.41 for three protons of the methyl group at C-4 (because of the anisotropic influence of the enone unit, the methyl group was shifted down-field). The two protons on C-3 appeared as an ABq-doublet e J=14.7 Hz. 3J=8.0 Hz, ' J-12.5 Hz) centred at 0 2.51 (OA = 2.4 and OB = 2.5). The complex signal at 0 3.5 1 was due to (j)-

proton on C-3a which coupled with the protons on C-

7a, C-3 and C-5 CJ=8 .0 Hz, 3J =12 .5 HZ, ' J=7.7 Hz. 4 J=1.7 Hz). The proton on C-7a appeared as a doublet at 05 .14 with a coupling constant ' J=7.7 Hz. The singlet at 0 5.79 was due to the proton of the dichloromethyl group at C-4. The olefinic protons at C-5 and C-6 gave an ABq. The proton at C-6 appeared at 0 6.31 CJ= I 0.3 Hz); another proton at C-5 gave a double-doublet at 0 6.60 CJ= I 0.3 Hz, 4J= 1.7 Hz).

The structure of a-methylene lactone 4 was determined on the basis of the following 'H NMR

data. The si nglet at 0 1.35 was assigned to three protons of the methyl group . The proton on C-3a

appeared at 0 4.0 as a broadened doublet due to

couplin g with the proton on C-7a and allyli c coupling with the two ne ighbouring proton on the exo-olefinic bond CJ=8 .8 Hz, 4J=2 Hz, 4J=3 Hz). The doublet at 0 5.4 was attributed to the proton on C-7a which

coupled with the proton on C-3a CJ=8 .8 Hz) . Two geminal olefinic protons on C-IO appeared as a characteristic broadened double-doublet at 0 5.75 and 6.30 due to allylic coupling with the ne ighbouring proton on C-3a e J=2 .5 Hz, 4J=2 Hz, 4J=3 Hz) . The two olefinic protons on C-5 and C-6 gave an ABq. The proton on C-6 appeared at 0 6 .15 ('J= 11.5 Hz) and the proton on c-5 at 06.5 ('J=11.5 Hz)25.

The IR spectrum exhibited absorption bands at 1765 (lactone carbonyl), and 1668, 1640 and 830 cm' l (double bond) ,

Bioassy

The biological activity of the lactone 4 was examined against some fungi and bacteria.

162 INDIAN J CHEM, SEC. B, FEBRUARY 1999

The results on the biochemical degradation of the lactone 4 have been reported earlier by Solujic and Sukdolak26 During tile- present investigation we found that lactone 4 had inhibitory influence on the metabilism of some fungi and bacteria which depended on the microbiological species, the nature of nutrient medium and concentration of lactone.

The aim of the present work was to examine the ability of the tested culture of fungi and bacteria to . biodegrade the lactone 4. Based on the chemical structure of degradation products, a plausible mechanisms has been suggested for lactone biodegradation.

In the first series of experiments the ability of degradation of lactone, using it as a constituent element of nutrient substrate at 2 mglmL concentration was investigated against the action of the fungi Aspergillus niger and Saccharomices cerevisiae fungi and the bacteria Agrobacterium tumefaciens and Bacillus mycoides.

In addition to the investigation of A. tumefaciens and B.mycoides with lactone as source of organic carbon two more microorganisms Pseudomonas glicinea and Pseudomonas Jluorescens were introduced into the second series of experiments. The lactone was added to the mineral medium of Czapek in concentration of ] mglmL and 7 mglmL.

Biodegradability of the lactone was examined in methylene chloride extract of the fermentation solution after the development of microorganism for 5 days. The results obtained were presented in Table I.

On the basis of the results obtained it can be concluded that both A. niger and B.mycoides degraded the lactone but at different rates. Degradation was slowlier in the case of A.niger.

Investigations carried out with lactone as the source of organic carbon revealed that in the case of A.tumefaciens, B.mycoides, P.glicinea and P. Fluorescens bacteria the consumption rates and the effect of degradation depended on the nature . of culture and lactone concentration.

Compounds A,B,C and D were identified on the basis of IR and NMR spectra29 .

Table I - Degradation Products of the lactone

Expt. Culture Lactone Starting Degradation No. mg/mL lacton products

RpO.24 Rp Lactone in nutrient medium

I. .A. niger 2 0.03,0.41 2. S.cerevisiae 2 3. B.mycoides 2 0.03,0.36,0.45 4. A. tumefaciens 2

Lactone as a source of carbon 5. A. tumefaciens I 0.43

7 6. B.mycoides I

7 OJJ9,O.47 0.13,0.60

7. P.glicinea 0.83,0.86

7 0. 17,0.66 0.78,0.87 0.92

8. P. j1uorescens I 7 0.48

The biodegradation mechanism 27-32 for the lactone also depended on microbiological species and was almost identical for A.tumefaciens, B.mycoides and P.jluorescens, but different for P.glicinea (cf. Scheme II).

10

SOLUJIC et aI. : MICROBIAL DEGR,ADA nON OF A SYNTHETIC BENZOFURANE TYPE LACTONE 163

Experimental Section

General. IR spectra were recorded using neat samples on a Perkin-Elmer grating spectrophotometer models 137 and 197. IH NMR spectra were recorded on a Varian FT 80 and 200 lemini spectrometers lusing TMS as internal standard (chemical shifts in 8, ppm: coupling constants J in Hz).

Trans- 3H, 3all, 7all- 4 -Dichloromethyl-4-me-thyl-2,7-benzofurandione 3. To a suspension of LTA (1.2 mmoles) in cold benzene (20 mL) and borontrifloride etherate (5 mL), a solution of compound 1 (590 mg, 1.2 mmoles) in benzene was added during 5 hr at room temperature. The reaction mixture was then poured into cold water (150 mL) and the solution obtained was filtered through Celite 577. The organic layer was separated .from aqueous layer, extracted with a saturated. solution of sodium bicarbonate and sodium chloride, and dried over anhydrous sodium sulfate. Removal of the sol vent afforded on oil which crystallized from ether, yield

71 %, mp 11 8 C; 'H NMR (CDCI1): 8 1.41 (s, 3H, CH,), 2.51 .(ABq, 2H, 2J-14 .71 Hz, 3J=8.06 Hz, 1J=12 .54 Hz), 3.5 1 (m, H, ' J= 8.06 Hz, JJ =12.54 Hz, ' J=7.7 3 Hz, 4J =1.79 Hz), 5 .. 14 (d, IH, ' J=7 .73 Hz), 5.79 (s, I H, CHClz), 6.31 (d, H , JJ= I 0.3 1 Hz), 6 .75 (dd, I H, ' J= to.3 1 Hz, 4J= 1.79 Hz); MS : mlz 248, 250 Anal. Calcd for C IOH, oO,CI 2: C 48. 19, H 4.02. Found: C, 48.56, H, 4.07%.

Trans- 3H, 3CJJ1, 7aH- 4 -Dichloromethyl-4-me-thyl- 3 -methylyliden- 2, 7 -benzofurandione 4. Sodium hyd ride dispersion in mineral oil (57%, 4.4 g 0.105 mole) was placed in a dry 250 mL three-necked flask to whIch an add itional funnel and mechanical stirrer were attac hed. The content was wa~hed three times wi th dry hexane and suspended in diethy l ether ( 100 mL, fres hl y distilled from LiAIH4) under

nitrogen . A mixture of y- Iactone (2.5 g, 0.0 1 mole) and ethyl for mate (0.74 g, 0 .0 I mole, dried over K2COJ and di still ed from P20 S) was slowly added to the stirred suspension, immedi ate ly following the addi tion of absolute ethanol (0.5 mL), at a rate that ma 'ntained a gentle reflux of the reaction so lvent. After stirring overnight the reac tion mixture was rapidly filtered under suction and the resulting solid material washed well with dry ether and dried ill vacuo to give the sodium salt of a-fo rmyl-y-Iactone as a li ght tan powder ( 1.95 g 65.5 %). The sod ium salt of a-formyl-y-I ac tone (1.45g 0.05 mole) and formaldehyde (200 mg 0.085 mole) were refl uxed in

THF under nitrogen atmosphere for 4 hr. Work-up

of the suspension gave the crude a -methylene-y-lactone which was obtained by filteration through a large funnel followed by removal of the solvent under reduced pressure. The crude reaction product was purified by passing through a 20 cm Kiselgel pad using dichloromethane as solvent. The solvent was removed under reduced pressure (in vacuo) and the residue repurified by the addition of 10 mL 6M hydrochloric acid and extraction thrice with 15 mL ether. The combined extracts were washed with a saturated sodium chloride solution dried over sodium sulfate anhydrous (Na2S04 anhyd.) and the sol vent

was evaporated in vacuo. The product a-methylene-y-Iactone was crystallized from ether-petrol, yield 730

mg (56%); "H NMR (CDCI,): 8 1.35 (s, 3H, CHJ), 4 .0 (brd. H, C,a-H, 3J=8 .8 Hz, 4J=2 Hz, 4J=3 Hz), 5.0 (d, H, C6 -H, ' J=8 .8 Hz) , 5 .75 (brd, H, C IO -H trans 2J=2 Hz, 4J=3 Hz), 6.30 (brd, H, C IO cis, 2J=2 Hz, 4J=2 Hz), 6.15 (d, H, C6-H, "J=11.2 Hz), 6.5 (d. H. C)-H, ' J= 11 .2 Hz): IR (KBr): 1765, (C=O, y-Iactone) , 1705, (c=O, enone), 1668, (C=C), 1649, (C:::CH}). 830 cm" C=CH2); MS: mlz 262/264 M+, Anal. Calcd for C II H IOO,C1 2: C, 50.59: H, 3.83. Found: C. 50.25 ; H,4.01 %.

Microorganisms: The strains Bacillus mvcoidc.', Agrohacterilll11 tumejaciells, Aspergillus lIiger, Saccharomyces cerevisiae, Pseudomonas fluo resc;ens and Pseudoll/onas glicill ea were taken from the collecti on of microorgani sms of the Faculty of Science, Department of Biology . Uni versity of Kragujevac.

Fermentation: A.niger and S. cerevisiae fun gi were inocu lated in a med ium of the foll owi ng composition : pepton I ( 15.0 g), yeast extrac t (5 .0 gl. dextrose ( 10.0 g), NaCI ( 10.0 g) and disti lled wate r (1000 mL). A suspension of the cu lture ( I mL) in 10 mL aqueeous substrate contai ning 2 mg of lac tone was incubated for five days at 22C.

B.mycoides and A.tumefaciens bacteri a were inocul ated into the substrate consisting of: a mixture ot pepton I greasy extrac t, NaCI and K2HPO.j (tota l weight 23.3 g) d issolved in 1000 mL of distilled water. A ~u spension of the cu lture ( I mL) into 10 mL of subst rate containing 2 mg of lactone was incubated fo r five days at 22C.

In the second series of investigation, in which lactone served as the source of organic carbon, a

164 INDIAN J CHEM, SEC. B, FEBRUARY 1999

modified nutrient substrate was used. B.mycoides, A. tumefaciens, P. glicinea and P.fluorescens were cultivated on a mineral substrate of the following composition: agar-agar (20.0 g), KNO, (2.0 g), MgS04:7H20 (0.8 g), FeS04. 7H20 (0.01 g) and NH4H2P04 (1 .0 g) dissolved in 1000 mL of distilled water (PH=7.0-7.2).

The lactone was added to the nutrient substrate in diffrerent concentrations: one of series of samples had I mg of lactone in 10 mL of substrate, whereas the other one had 7 mg of lactone in 10 mL of substrate.

After the separation of mycelium, half (5 mL) of the culture was extracted with the same volume (5 mL) of CH2CI2 for the purpose of identifying the start ing lactone, the number and the chemical structure of degraded lactone products.

The extract, thus obtained, was dried over anhydrous Na2S04, filtered, concentrated, and analyzed by TLC on silica-gel layer using hexane-acetone (6:4) as irrigant. Chromatographic spots were developed and detected in iodine vapours or by sprinkling the chromatogram with con. H2S04 and heating for 30 min. at 150C. Rr values were determined in comparison to the standard, starting lactone.

Identification of extracellular metabolic degra-dation products of synthetic lactone was performed by preparative TLC on silica-gel using hexane-acetone as the solvent system. Spot fronts of unknown compounds were marked by comparing with a chromatogram obtained under the same conditions when the spots were visible after developing the chromatogram with H2S04. The portion of silica gel layer corresponding to an unknown compound was removed from the plate and extracted with CH2C12. After drying the extract with Na2S04 (anhyd.), the solvent was removed and the crude metabolic mixture analyzed by IR and 'H NMR data and compared with literature data22 .

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22 Pretsch E, Seibi 1 & Simon W, Tabellen zur Strukturaufkarung Organischer Verbindungen mit Spektroskopischen Methoden, (Springer Verl ag, Berlin) 1987 .

23 'H NMR (CDCI , ) - Compound A: two protons (CH2CI) at C-4, ABq, 8 3.5 ; C-2 and C-3 two protons, ABq at 8 6.58 (1=7.3 Hz); exocyclic methylene, two protons, brd at 85.95 (trans-COOH) , 8 5.82 (cis-COO H), 2J=1.7 Hz, 41=0.2 Hz; COOH, brs at 89.15 (HDO); IR (KBr): 1695, i. 680, (C=O), 3630 cm1 (OH) . Compound B: two singlets for the two methyl group at C-4 (8 1.38 and 1.3 1, six protons). Compound C : two protons (CH2 OH) conversion into 8-lac ton , ABq at 8 3.95. Compound D: three protons of the methyl group (C H,) at C4, d at 8 1.38 (J= 8.2 Hz), one proton at C-4, dq at 87 (1=11.3 and 1=8.2 Hz). Compou nd E: DC NMR: 8 183.9 (C=O of 10-COOH), 19 1. 2 (C=O at C-I), 169.3 (C=O of 8-COOH).

SOLUJIC et al. : MICROBIAL DEGRADATION OF A SYNTHETIC BENZOFURANE TYPE LACTONE 165

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