Chem. Res. Toxicol. 1993,6, 46-49
46
Synthesis and Toxicity to Mammalian Cells of the Carrot Dihydroisocoumarins Stefan0 Superchi,t Dario Phi,+ Piero Salvadori,**tFlavia Marinelli,t Giuseppe Rainaldi,! Ugo Zanelli,§ and Vittoria Nuti-Ronchis C.N.R. Centro S t u d i Macromolecole Stereordinate e d Otticamente Attive, Dipartimento di Chimica e Chimica Industriale, Universith d i Pisa, Via Risorgimento, 35, 56126 Pisa, Italy, Scuola Normale Superiore, Piazza dei Cavalieri, 56100 Pisa, Italy, a n d C.N.R. Istituto d i Mutagenesi e Differenziamento, Via Svezia, 10, 56100 Pisa, Italy Received J u n e 22, 1992
The dihydroisocoumarins (f)-6-methoxy-8-hydroxy-3-methyl-3,4-d~ydro~~oumarin (l),(&)6,8-dihydroxy-3-methyl-3,4-dihydroisocoumarin (2), and (&)-6,8-dimethoxy-3-methyl-3,4-dihydroisocoumarin (3) were synthesized with high yields via metalation of o-methylbenzamides, The toxicity of these compounds and that of (-)-l extracted from carrot cells were tested, in vitro, on Chinese hamster cells. The toxicity was determined according to the presence or absence of a hydroxyl group in the peri position of the lactonic carbonyl group and according to the stereochemistry of the dihydroisocoumarin. Introduction Isocoumarins and 3,4-dihydroisocoumarins are substances widely present in nature as secondary metabolites of plants and fungi. Many show interesting biological properties (1, 2) and have potential commercial use. During our study on the potential biotechnological application of cultured carrot cells for antibiotic production (3), our interest was attracted to the phytoalexin (4, 5 ) 6-methoxy-&hydroxy-3-methy1-3,4-dihy&oisocoumarin(I) (Figure 11, showingantibioticproperties, and i h immediate biosynthetic precursor, 6,&dihydroxy-3-methyl-3,4-dihydroisocoumarin ( 2 ) (6). Both are produced by roots of D a u c u s carota after infection with several fungi or exposure to physical and chemical agents of stress (7-10). A research project is in progress in order to elucidate the biosynthetic pathway leading to the production of 1 and 2 in carrot cells and the nature of the biological activity of these 3,4-dihydroisocoumarins and related compounds. Therefore, our attention focused on the study of reliable synthetic methods to generate sufficient material with which to carry out enzymology studies and biological activity assays. In this paper, we describe the development of an efficient method for the synthesis of (f)-1, (f)-2, and their methyl ether (f)-3. The comparison of the toxic effect on mammalian cells of both the synthetic compounds and the natural enantiomer (-)-l and a preliminary study on the correlation between the biological activity, the nature of aromatic substitution, and the stereochemistry of these bioactive molecules are also described. Experimental Section General Procedures. Melting points were measured with a Kofler hot-stage apparatus (Reichert, Austria) and were uncorrected. IR and UV spectra were recorded on a Perkin-Elmer (Norwalk, CT) 1750 and on a JASCO (Tokyo, Japan) UVIDEC t C.N.R. 1 Scuola 8 C.N.R.
Macromolecole, UniversitA Pisa. Normale Superiore. Mutagenesi. The quantities of 1 and 2 available from infected carrot roots are too small to carry out extensive studies.
R’O
Q
(1)
R ’ = H, R2= CH3
(2)
R 1 = R2= H
(3)
R1=
R2=
CH3
Figure 1. Structures of dihydroisocoumarins 1-3. 710 spectrophotometer, respectively. NMR spectra were obtained on a Varian (Palo Alto, CA) Gemini 200 or XL 300 spectrometer using tetramethylsilane as internal standard. Mass spectra were carried out by electron impact on a VG 70-70E spectrometer. Optical rotatory power (a)was determined on a JASCO Dip 360 automatic polarimeter. Silica gel 60 (70-230 mesh) and 60 F-254 (0.2 mm) (Merck, Hohenbrunn, Germany) were used for column and TLC,2 respectively. Metalations were carried out using syringe-septum cap techniques under nitrogen atmosphere. sec-BuLi (Aldrich, Milwaukee, WI) was a 1.4 M solution in cyclohexane/isooctane, and n-BuLi (Aldrich) was a 1.6 M solution in hexane; the exact titer of both was determined by titration using 2,5-dimethoxybenzyl alcohol (11). Diisopropylamine (Fluka, Buchs, Switzerland) and TMEDA (Aldrich) were distilled from NaH and CaHz, respectively, and stored over 4-A molecular sieves. THF was dried by distillation from LiAlH. immediately before use. (A) N,IV-Diethyl-2,4-dimethoxybenzamide (4). Neat SOClz (48 mL, 658 mmol) was added to 2,4-dimethoxybenzoic acid (30 g, 164 mmol), and the resulting mixture was heated under reflux for 6 h. After removing excess SOC12, the resulting solid residue was redissolved in T H F (100 mL) and added dropwise, a t 0 OC, to diethylamine (34 mL, 327 mmol). The mixture was heated under reflux with overnight stirring and then hydrolyzed. T H F was removed under vacuum, the residue dissolved in CHCls, and the organic layer treated in sequence with 10% aqueous HC1, 10 96 aqueous Na2C03,and water and dried over Na2SOI. After concentration of the solution, distillation at 160-162 OC/O.7 Torr yielded amide (4)(34.5 g, 88%)as a slightly yellow oil. Anal. Abbreviations: TLC, thin-layer chromatography; TMEDA, NAN,”-
tetramethylethylenediamine;THF, tetrahydrofuran; LDA, lithium di-
isopropylamide; DMSO, dimethyl sulfoxide;set-BuLi, set-butyllithium; n-BuLi, n-butyllithium.
0893-228x/93/2706-0046$04.00/00 1993 American Chemical Society
Toxicity of Carrot Dihydroisocoumarins
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 47
water and dried over Na2S04. Evaporation of solvent gave a residue which, after chromatography with C6H6/CH30H (100:3), yielded 99 mg (65%) of 1 as white needle-shaped crystals, mp 93-96 "C [lit. (12) 93 "C]. Anal. Found: C, 63.62; H, 5.93. q,J7.1Hz,NCH2CH3),3.55(2H,brq,J7.1Hz,NCH2CH3),3.79 CllH1204 requires C, 63.46; H, 5.77. ,v (KBr) 3019 (OH), 1657, (3H,s,OCH3),3.81(3H,s,OCH3),6.46(1 H,d,J2.4Hz,aromatic), (CH30H) 216 nm; lH NMR (200 MHz; 6.48(1H,dd,J2.4and8.2Hz,aromatic),7.12(1H,d,J8.2Hz, 1629 (CO) cm-l; A,, CDC13) 6 1.51 (3 H, d, 56.25 Hz, CHCH3), 2.87 (2 H, d, J 6 . 8 Hz, aromatic). ArCH3CH), 3.82 (3 H, s, OCH3), 4.68 (1 H, m, CH2CHCH3), 6.25 (B)N,N-Diethyl-2,4-dimethoxy-6-methylbenzamide (5). (1H, d, J 2.3 Hz, aromatic), 6.37 (1 H, d, J 2.3 Hz, aromatic), A stirred solution of sec-BuLi (8.9 mL, 12.4 mmol) and TMEDA 11.25 (1 H, s, ArOH); m/z 208 (M+, 100) and 190 (M+ - 18). (1.87 mL, 12.4 mmol) in dry T H F (60 mL) was prepared a t -78 (F)(*)-6,8-Dihydroxy-3-methyl-3,4-dihydroisocoumarin "C and treated with amide (4) (2.5 g, 10.55 mmol) in T H F (25 (2). A stirred solution of methyl ether 3 (1.5 g, 6.75 mmol) in mL) by slow injection. The solution was stirred for 30 min, and CHzClz(70 mL) a t -78 "C was treated with a solution of BBr3 dry methyl iodide (3.52mL, 56.5 mmol) was injected. The mixture (6 mL, 63.5 mmol) in CH2C12 (50 mL) by dropwise addition. The was allowed to warm up to room temperature with overnight mixture was allowed to reach room temperature with overnight stirring. The mixture was then hydrolyzed, and T H F was stirring and then was treated with 10% aqueous NaHC03 and subsequently evaporated. The residue was dissolved in CH2C12 diluted with CHzClz (100 mL). The organic layer was treated and the organic layer washed with 10% aqueous HCl and water with 10 % aqueous NaOH; the aqueous extract was acidified with and then dried over Na2S04. Evaporation of the solvent gave 10% aqueous HCl and extracted with CHC13. The chloroform amide (5) (2.6 g, 97%) as a viscous oil. Anal. Found: C, 67.05; layer was then washed with water and dried over Na2S04. The H, 8.52; N, 5.75. Cl4HZ103Nrequires C, 66.93; H, 8.37; N, 5.58. residue, obtained after evaporation of solvent, was chromato1H NMR (200 MHz; CDC13) 6 1.03 (3 H, t, 5 7 . 1 Hz, NCHzCHs), graphed with C6H6/CH30H(10:l) and gave 900 mg (68%) of 2. 1.23 (3 H, t, J 7.1 Hz NCHZCH~), 2.22 (3 H, s, ArCH3), 3.12 (2 Recrystallization from acetone/hexane gave an analytical sample H, q, NCH2CH3, J 7.1 Hz), 3.30-3.55 (1H, m, NCH2CH3), 3.60of 2 as yellow prisms, mp 205-210 "C [lit. (13, 14) 209-211 "C]. 3.76 (3 H, s,OCH3),3.79 (3 H, s, oc&), 3.90 (1H, m, NCHZCH~), Anal. Found: C, 62.05; H, 5.30. C10H1004requires C, 61.85; H, 6.30(1H,d,J2.4Hz,aromatic),6.34(1H , d , J2.4Hz,aromatic). (CH35.15. ,v (KBr) 3225 br (OH), 1657,1630 (CO) cm-l; A,, (C) N,N-Diethyl-2,4-dimethoxy-6-(2'-hydroxypropyl)OH) 216 nm; lH NMR (200 MHz; acetone-&) 6 1.45 (3 H, d, J benzamide (6). A solution of diisopropylamine (0.89 mL, 6.38 6.3 Hz, CHCH3), 2.84 (1H, dd, J 16.4 and 10.6 Hz, ArCHzCH), mmol) in dry T H F (50 mL) was treated, under nitrogen a t 0 "C, 2.97 (1 H, dd, J 16.4 and 4 Hz, ArCHZCH), 4.70 (1 H, m, with n-BuLi (1.6 M in hexane; 4 mL, 6.38mmol) and the mixture CH2CHCH3), 6.27 (1 H, d, J 2.3 Hz, aromatic), 6.29 (1 H, d, J stirred for 15 min a t room temperature. After cooling to -78 "C, 2.3 Hz, aromatic), 9.44 (1H, br s, ArOH), 11.30 (1H, s, ArOH); a solution of 5 (1g, 3.98 mmol) in T H F (7.5 mL) was added by mlz 194 (M+, 100) and 176 (M+ - 18). syringe injection. The deep red solution was stirred for 1h and ( G ) (-)-6-Methoxy-8-hydroxy-3-methyl-3,4-dihydroiso(0.28 g, 6.38 mmol). The treated with an excess of acetaldehyde coumarin (1). The natural enantiomer (-)-lwas isolated from mixture was stirred a t -78 "C for 5 min and a t room temperature carrot roots infected by Fusarium solani and purified by TLC for 15hand then hydrolyzed. T H F was subsequently evaporated and acid-base extraction as previously reported (7). The and the residue dissolved in CH2C12. The organic layer was procedure gave a sample of (-)-l,whose spectroscopic charactreated with 10% aqueous HCl, washed with water, and dried terization completely agreed with that of the synthetic sample over Na2S04. Evaporation of solvent afforded an oil which, after (*)-l. chromatography (EtOAcCHC13, l:l), yielded 170 mg of 5 and Toxicity Assay of (-)-1, (*)-1, (*)-2,a n d (f)-3 t o Chinese 900 mg (77%) of amide alcohol 6. Anal. Found: C, 65.26; H, Hamster Cells. Chinese hamster VP 79-AP4 cells, isolated and 8.62; N, 4.79. C1&O4N requires c, 65.08; H, 8.47; N, 4.74. vmax maintained by the procedure of Colella et al. (15),were treated (KBr) 3373 br; 1603 (CO amide) cm-'; lH NMR (200MH2, CDC13) with trypsin, counted, and plated (250 cells per 6-cm diameter 6 1.00-1.10 (3 H, m, NCH~CHS),1.20-1.35 (6 H, m, CH2CH3 + Petri dish) in DMEM Gibco medium ( 1 9 ,containing 100 IU/mL CHCHs), 2.3-2.8 (2 H, m, ArCHzCH), 3.03-3.31 (2 H, m, NCH2penicillin, 100 wg/mL streptomycin, and 5% fetal calf serum. CH3),3.40-4.0(3H,m,NCH,CH,+ CHOH),3.77 (3 H,s,0CH3), After 6 h of growth, the growth medium was replaced with fresh 3.82 (3 H, s, OCH3), 4.08-4.25 (1H, m, CHCH3), 6.30-6.45 (2 H, medium containing appropriate amounts of isocoumarins, prem, aromatic). (D)(*)-6,8-Dimethoxy-3-methyl-3,4-dihydroisooumarin viously dissolved in DMSO. The final concentration of DMSO was adjusted to 0.5 % v/v in all conditions, and a control containing (3). The amide alcohol 6 (580 mg) was treated with a mixture only 0.5% vlv DMSO was used. After incubation of 7 days, of 50% aqueous NaOH (20 mL) and EtOH (20 mL) and heated colonies were colored with methylene blue vital stain and counted. under reflux for 36 h. The reaction mixture was evaporated to Results are presented as a mean from a t least three replicates dryness, acidified with concentrated HCl a t 0 "C, and extracted and data were expressed as the percentage of the number of with ethyl acetate. The organic extract was washed with water, colonies grown compared to the DMSO control. driedover Na2S04,and concentrated to give an oily residue which, after recrystallization from diethyl ether/hexane, yielded 410 mg (88%)of 3 as white prisms, mp 102-104 "C [lit. (12) mp 103 "C]. Results and Discussion Anal. Found: C, 64.98; H, 6.45. ClzH1404 requires C, 64.86; H, Synthesis of Racemic Dihydroisocoumarins. Pre6.31. vmaX(KBr) 3093,1708 (CO), 1605,1582 cm-l; A,, (CH30H) viously reported synthetic methods for 1-3 usually proceed 216 nm; lH NMR (200 MHz; CDC13) 6 1.47 (3 H, d, J 6.4 Hz, CHCH3), 2.78 (1 H, dd, J 16 and 4.6 Hz, ArCH2CH),2.89 (1 H, via one of t h e following: ortho-lithiated secondary benzdd, J 16 and 10 Hz, ArCHzCH), 3.87 (3 H, s, OCH3), 4.52 (1H, amides (12),o-methylbenzoic esters (16),homophthalic m, CHZCHCHS), 6.31 (1 H, d, J 2.3 Hz, aromatic), 6.41 (1H, d, anhydrides (17),indanones (13,141, or indandiones (18). J 2.3 Hz, aromatic); mlz 222 (M+, 100) and 205 (M+ - 17). However, all of these approaches present disadvantages. (E) (~)-6-Methoxy-8-hydroxy-3-methyl-3,4-dihydroiso- T h e reaction of ortho-lithiated secondary benzamides with coumarin (1). To a stirred solution of methyl ether 3 (163 mg, propylene oxide gives poor yields. Syntheses via indanones 0.734 mmol) in dry T H F (40 mL) was added 500 mg of AlC13 and homophthalic anhydrides comprise m a n y steps. under nitrogen. The mixture was heated under reflux for 12 h Precursors such a s substituted o-methylbenzoic esters a r e and the solvent evaporated under reduced pressure. The residue difficult t o prepare and, if commercially available, very was treated with 10% aqueous HCl and extracted with CHzC12. expensive. Moreover, t h e reaction of their o-toluate anions The organic layer was extracted with 10% aqueous NaOH; the with acetaldehyde gives dihydroisocoumarins as well as aqueous solution was acidified with 10% aqueous HCl and then extracted with CHC13. The chloroform solution was washed with self-condensation byproducts. Found: C, 65.95; H, 8.22; N, 6.02. C13N1903N requires C, 65.82; H, 8.02; N, 5.91 76. 1H NMR (200 MHz; CDC13) 6 1.03 (3 H, t J 7.1 Hz, NCH~CHS), 1.23 (3 H, t, J 7.1 Hz, NCHZCH~), 3.16 (2 H,
48 Chem. Res. Toxicol., Vol. 6, No. 1, 1993
Superchi et al.
Scheme Ia CONEt2
Me
CONEt,
Me (1)
Me
Me
(3)
I
I'"
Me
(2)
Reagents and conditions: (a) sec-BuLi, TMEDA, THF, -78
OC;
(b) CH& (c) LDA, CH3CHO; (d) H30+;(e) NaOH, HzO, EtOH, 78 "C; (0 AlC13, THF, 65 OC; (g) BBr3, CHzClz, -78 "C.
The synthesis of 1-3 (see Scheme I) based on a modification and extension of the o-methyl tertiary benzamide approach used by Watanabe et al. (19)looked very promising. These authors carried out the reaction of such amides, lithiated on the benzylic position, with aromatic aldehydes, obtaining several 3-aryl-3,4-dihydroisocoumarins. We extended the same reaction to an aliphatic aldehyde (20),as acetaldehyde, in order to achieve 3-alkyl-3,4-dihydroisocoumarins as the methyl ether 3, which is the precursor of 1 and 2. Amide 4,synthetized from the corresponding benzoic acid, was regiochemically ortho-lithiated by sec-BuLi, in the presence of TMEDA at -78 OC, followingthe procedure of Beak and Brown (21). Methyl iodide was added to the resulting yellow solution of lithiated amide 4,obtaining a white suspension. The workup afforded o-toluamide 5 in 97 7% yield. Amide 5 was further lithiated on the methyl group with LDA in THF at -78 OC, yielding a red solution which, after quenching with acetaldehyde, gave amide alcohol 6 (77% yield). Since hydrolysis of N,N-diethylbenzamides takes place in harsh conditions and only when it is joined with an intramolecular cyclization, we performed hydrolysis of amide alcohol 6 in strong basic conditions, heating under reflux in aqueous 50% NaOH/EtOH solution for 36 h. The procedure afforded the dihydroisocoumarin 3 in high yield (88%). Our synthesis gave the methyl ether 3 in three steps, from the starting compound 4, and with an overall yield of 65%, which is almost twice the yield obtained in the earlier procedures. As a matter of fact, Carpenter et al. (16) prepared 3 in three steps and 34% overall yield starting from triacetic lactone methyl ether, while Henderson and Hill (17) obtained 3 from 2,4dimethoxy-6-methylbenzoic acid in four steps and 35 % yield. Moreover, the method of Slates et al. (18) allows one to prepare 3 in 53% yield, starting from 5,7dimethoxyindanone, but this procedure is longer than the others, comprising six steps. Afterward, methyl ether 3 was selectively demethylated by heating under reflux with AlCl3 in THF for 24 h. Dihydroisocoumarin 1 was isolated in 65% yield. The use of THF as solvent instead of ethyl ether (12) allowed us to completely solubilizethe product 3 and reach a higher reaction temperature, thus increasing the conversion considerably. Complete demethylation Of 3 was performed with BBr3 in CH2C12 at -78 "C, obtaining, after workup, the dihydroisocoumarin 2 in 68% yield. Our procedure is very efficient, providing 1-3 in high yield, is comparatively easy to perform, and presents a potential versatility toward the synthesis of dihydroiso-
2o
1
0 0,O
\ 0,2
0,4
O,b
0,8
1,0
1 2
Dihydroisocoumarin (mM) Figure 2. Number of colonies of V79-AP4 Chinese hamster cells grown in different concentrations of (*)-1 (A),(f)-2 (m), and (k1-3 (0).
coumarins with different substitutions either on the aromatic or on the lactonic ring. Toxicity of Dihydroisocoumarins (*)-l,(f)-2, and (*)-3and of the Natural Enantiomer (-)-1 toChinese Hamster Cells. The biological activity of phytoalexins has been extensively studied in vitro using different types of bioassays. In general, these molecules are toxic to fungi, bacteria, plant cells, and animal cells (4, 22). Recently, it has also been suggested that some phytoalexins are carcinogenic (23). Carrot phytoalexin (+l is known to inhibit the growth of several fungi and bacteria and to markedly reduce viability of carrot cells in the range 10"L10-5 M (8,24,25). Nevertheless, very little is known about its biological activity on animal cells (7). We report the results on the toxicity of the structurally related isocoumarins (*)-l,(*)-2, and (f)-3 and of (-)-l to VP79-AP4 Chinese hamster cells in vitro (15). Increasing amounts of the dihydroisocoumarins were added to colonies of Chinese hamster cells, and the percentage of living colonies compared to the control was plotted. In Figure 2, toxic effects of (+l, (A1-2, and (*)-3 are compared: (f1-l was more toxic than (*)-2, and (*)-3 showed practically no activity up to 1 mM. In fact, treatment with 1 mM (&)-1did not allow the growth of any colony. The number of colonies was, indeed, reduced to 45 % and 91% of the control after treatment with 1mM (*)-2 and (&)-3, respectively. These results show that 0-methylation of the hydroxyl group in the 6 position confers major toxicity to (k1-1 compared to (*I-2. Nevertheless, 0-methylation of the hydroxyl group at the 8 position in the isocoumarin almost completely inhibits the biological activity, demonstrating that toxicity is related to the presence of a hydroxyl group in the peri position of the lactonic carbonyl. NMR and IR spectra of 1 and 2 revealed that these two groups are linked, as expected, by an intramolecular hydrogen bond. The former revealed that the protons of the phenolic group in the 8 position gave a narrow peak at 11.25 and 11.30 ppm, respectively. The proton of the phenolic group in the 6 position of 2, free from intramo-
Toxicity of Carrot Dihydroisocoumarins lecular bonding, gave a wide signal at 9.44 ppm. Moreover, in the IR spectrum of 1, the OH stretching at 3019 cm-l is much less intense than the signal corresponding to the free phenolic group in the IR spectrum of 2. According to some authors (26)the peri position of the hydroxyl and the carbonyl group in 1allowsthe moleculeto bind divalent cations such as Mg2+and Ca2+,inhibiting the action of the enzymes activated by these metals. We compared the toxicity of W - 1and of (-1-1 of natural origin in order to have preliminary information on the effects of stereochemistry on biological activity. The (3enantiomer was more toxic than the racemic preparation, their ED503 values being 0.46 and 0.66 mM, respectively. At at 1mM of either (-1-1 or (&)-1,no colony was detected. Therefore, even for the carrot phytoalexins as well as for several natural and pharmacological compounds ( 2 3 ,the presence of a chiral center in the molecule influences the biological activity. Further studies, with the availability of the (+)-lenantiomer, are necessary to establish a true correlation between activity and stereochemistry. From these resulta we can conclude that the natural (+l carrot phytoalexin is more toxic than the synthetic (&)-l,(*)-2, and (&)-3dihydroisocoumarins on Chinese hamster cells. The toxic effect of (+l on this animal cell line (E& = 0.46 mM) is lower than on microorganisms and plant cells (EDSO= 0.04-0.05 mM) (9, 10,24, 25). Even if further and more extended assays must be carried out, the low absolute toxicity level of (-1-1 on Chinese hamster cells suggeststhat this naturally occurring carrot metabolite does not have significant cytotoxic activity on animal cells. In conclusion, we reported an efficient synthetic method for the preparation of the dihydroisocoumarins 1-3. This procedure allowed us to obtain these products in large amounts and to carry out biological activity assays. From these assays we found that the presence of a hydroxyl group in the peri position of the lactonic carbonyl group strictly influences toxicity. Furthermore, we have reported interesting preliminary results on the correlation between biological activity and stereochemistry of phytoalexin 1.
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