Synthesis and structure-activity relationships of antifungal crassinervic acid analogs

Article abstract of DOI:10.1002/cbdv.201100288

A series of analogs of the natural antifungal compound crassinervic acid, a constituent of Piper crassinervium, were synthesized to gain insight into the most relevant structural features affecting the activity of the parent molecule. Most compounds were

Full text of DOI:10.1002/cbdv.201100288

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
41
Synthesis and StructureꢀActivity Relationships of Antifungal Crassinervic
Acid Analogs
by Jyotsna N. Chakora), Loana Mussoa), Paola Sardib), and Sabrina Dallavalle*a)
a
`
) DISMA, Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, via Celoria 2,
IT-20133 Milano (phone: þ39-02-50316818; fax: þ39-02-50316801; e-mail: sabrina.dallavalle@unimi.it)
b
`
) Dipartimento di Protezione dei Sistemi Agroalimentare e Urbano e Valorizzazione delle Biodiversita,
`
Universita degli Studi di Milano, via Celoria 2, IT-20133 Milano
A series of analogs of the natural antifungal compound crassinervic acid, a constituent of Piper
crassinervium, were synthesized to gain insight into the most relevant structural features affecting the
activity of the parent molecule. Most compounds were prepared by aldol reaction of methyl 3-acetyl-4-
hydroxybenzoate with a series of ketones, followed by reduction, hydrolysis, and oxidation. The
antifungal activities of crassinervic acid and its analogs was assessed against Cladosporium cladospor-
ioides by using the method of bioautography. The bioassay results allowed us to depict structureꢀactivity
relationships for this class of compounds.
Introduction. – The Piper species are an abundant source of natural antifungal
compounds [1]. A recent investigation [2] of the components of Piper crassinervium
from Brazil led to the isolation and the assessment of the antifungal activity against
some Cladosporium species of a compound to which the structure of (ꢀ)-crassinervic
acid (1; configuration unknown) was assigned.
More recently, we undertook the total synthesis of the structure assigned to
crassinervic acid, and prepared both the racemic, 1, and the levorotatory form, (ꢀ)-1, to
which the (S) configuration at C(3’) was assigned [3]. The convergent character of the
synthesis of 1 made possible its easy application for the preparation of analogs. Here,
we report on the synthesis of a series of analogs of 1 and the assessment of their
antifungal activities against Cladosporium cladosporioides. As the antifungal activities
of the racemic and the optically active form of 1 were almost identical (see the Table),
we undertook the synthesis of the analogs in racemic form.
Results and Discussion. – To investigate the importance of the side chain of 1 for
activity, we prepared the saturated derivative 7c, and two analogs with a shorter, i.e., 7b,
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table. Antifungal Activity [mg] of Crassinervic Acid (1) and its Analogues
Compound
Antifungal activity [mg]
against C. cladosporioides
Compound
Antifungal activity [mg]
against C. cladosporioides
(ꢀ)-1
1
10.0
10.0
–
7b
7c
7d
8
20.0
10.0
20.0
–
4a
4c
–
5a
5c
6a
6c
–
–
50.0
–
9
14
15
10.0
30.0
50.0
and longer, i.e., 7d, chain (Scheme 1). The three analogs were prepared by a sequence
of reactions involving aldol reaction [4] of methyl 3-acetyl-4-hydroxybenzoate (2) with
the appropriate ketone 3a–3d, followed by NaBH4 reduction of the keto group, LiOH
hydrolysis of the esters 5a–5d, and re-oxidation of the secondary alcohols 6a–6d to the
ketones 7b–7d. This reductionꢀoxidation cycle was necessary because of the sensitivity
of the aldol products 4a–4d both to basic (retro-aldol reaction) or acidic (dehydration)
conditions required for the hydrolysis of the esters (Scheme 1).
Scheme 1
a) Lithium diisopropylamide (LDA), THF, ꢀ788, 3 h; 56% for 4a, 82% for 4b, 73% for 4c, 79% for 4d.
b) NaBH4, MeOH, 08 to r.t., 2 h; 83% for 5a, 75% for 5b, 80% for 5c, 82% for 5d. c) LiOH·H2O, THF/
H2O, reflux, 9 h; 66% for 6a, 100% for 6b, 96% for 6c, 67% for 6d. d) 2-Iodoxybenzoic acid (IBX),
DMSO, r.t., 3 h; 71% for 1, 68% for 7b, 69% for 7c, 70% for 7d.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
43
The influence of the other functional groups of 1 on the activity was studied by
examining the esters 4, and 8a and 8b, two geometrical isomers formed by treatment of
compound 4a with SOCl2/Et3N in CH2Cl2 (Scheme 2). The configurations of the side
chains of 8a and 8b (Scheme 2) were established on the basis of the 1H-NMR spectra.
Attempt to hydrolyze the mixture of 8a and 8b led to an intramolecular Michael
reaction, with the formation of the chromanone 9 [3].
Scheme 2
a) SOCl2/Et3N, CH2Cl2, 08, 2 h; 80%. b) NaOH/H2O, r.t., overnight; 100%.
Similarly, the effect of the phenolic OH was checked by comparing the activity of 1
with that of its methyl ether 14. This compound was obtained via a sequence of
reactions similar to those used for the synthesis of the corresponding phenol
(Scheme 3).
Scheme 3
a) LDA, THF, ꢀ788, 3 h; 56%. b) NaBH4, MeOH, 08 to r.t., 2 h; 83%. c) LiOH·H2O, THF/H2O, reflux,
9 h; 99%. d) IBX, DMSO, r.t., 3 h; 70%.
Comparison of the activities of 1 and 7c with those of the corresponding alcohols 6a
and 6c provided information about the effect of the keto group. As both the phenolic

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
OH and the COOH functions seem to be important for the antifungal activity, we
tested also the isomer 15 [3] of 1, as it is known that salicylic acids may have antifungal
activity [5].
The antifungal activity of this series of analogues against Cladosporium cladospor-
ioides was assessed by the method of bioautography [1d][6][7] (Table).
The antifungal test results of all the tested compounds and their structural
characteristics have revealed a structureꢀactivity relationship which is summarized as
follows:
1. Both enantiomerically pure and racemic crassinervic acid showed almost the
same antifungal activity. This means that chirality is not crucial for activity.
2. Saturation of the C¼C of the side chain (i.e., 7c), and replacement with saturated
chains of different length and substitution (i.e., 7b and 7d) clearly demonstrated that
the C¼C in the side chain is not crucial for antifungal activity.
3. Reduction of the keto C¼O group caused a strong decrease in antifungal activity
(cf. 6a and 6c). Protection of the benzoic acid group as an ester (i.e., 4a, 4c, 5a, 5c, and
8) resulted in complete loss of activity, implying that the benzoic acid moiety is essential
for good antifungal activity.
4. Protection of phenolic OH as methyl ether (i.e., 14) caused a certain decrease in
activity.
5. The dehydrated aldol adducts 8a and 8b showed loss of activity, but this may be
due to the esterification of the acid group. Unfortunately, it was not possible to obtain
the corresponding acids, as, during hydrolysis, cyclization to chromanone 9 occurred.
6. The 4-hydroxybenzoic acid structure is important for activity (i.e., 15 vs. 1).
Compound 9 showed activity comparable with that of crassinervic acid. This is an
interesting result because, as we suggested in our previous work [3], in vivo H2O
elimination at 3’ position of crassinervic acid, followed by intramolecular Michael
addition, could afford chromanone 9. In fact, a biosynthetically related bioactive
compound has recently been isolated from a different Piper species (Piper obliquum)
by Sterner and co-workers [8].
Conclusions. – In this work, a series of crassinervic acid analogs were synthesized
and assayed for structureꢀactivity relationship studies. The bioassay results clearly
indicate that the structure of a benzoic acid with an O function in para-position is
required. Moreover, a lipophilic acyl moiety in meta-position with respect to the
COOH group is crucial for the antifungal activity in this series of compounds. Finally,
the interesting biological activity exhibited by compound 9 made 2-substituted 4-
oxochroman-6-carboxylic acids worth of further investigation as antifungal compounds.
The authors thank the University of Milan (FIRST Funds) for providing financial support.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
45
Experimental Part
General. Solvents were routinely distilled prior to use; anh. THF and Et2O were obtained by
distillation from sodiumꢀbenzophenone ketyl; dry CH2Cl2 was obtained by distillation from P2O5. All
reactions requiring anh. conditions were performed under a positive N2 flow, and all glassware was oven-
dried and/or flame dried. All reagents and solvents were reagent grade or were purified by standard
methods before use. M.p.: in open capillaries on a Bꢀchi melting-point apparatus; uncorrected. Column
chromatography (CC): silica gel (SiO2; 230–400 mesh; Merck). TLC: on silica gel plates (Merck 60 F254).
1H-NMR Spectra: at 300 MHz with a Bruker instrument; chemical shifts (d) and coupling constants (J) in
ppm and Hz, resp.
Compounds 1, 4a–6a, 8a and 8b, 9, and 15 were prepared as described in [3].
General Procedure for the Synthesis of Compounds 4b–4d and 11. Under Ar, a soln. of LDA was
prepared by adding a 2.7m soln. of BuLi in heptane (0.358 ml, 0.97 mmol) to a soln. of iPr2NH (0.136 ml,
0.97 mmol) in 3 ml of THF under stirring at 08. After 30 min, the soln. was cooled to ꢀ788. A soln. of
methyl 3-acetyl-4-hydroxybenzoate (2) or methyl 3-acetyl-4-methoxybenzoate (10; 0.39 mmol) in THF
(1 ml) was then slowly added over 10 min. The resulting soln. was stirred for 1 h at ꢀ788 (to secure the
complete formation of the corresponding enolate) prior to slow addition of the appropriate ketone
(0.97 mmol). Stirring was continued for 3 h at ꢀ788, and then the reaction was quenched with a sat. aq.
NH4Cl soln. The mixture was allowed to warm to r.t. The layers were separated, and the aq. layer was
extracted with Et2O (3ꢁ5 ml). The combined org. layer was dried (anh. Na2SO4), filtered, and
concentrated. The residue was purified by flash CC (AcOEt/hexane 15 :85) to afford the aldol product.
Methyl 4-Hydroxy-3-(3-hydroxy-3,5-dimethylhexanoyl)benzoate (4b). Yield 82%. Rf (AcOEt/
1
hexane 25 :35) 0.63. H-NMR (CDCl3): 12.5 (s, 1 H); 8.48 (d, J¼1.7, 1 H); 8.13 (dd, J ¼ 1.7, 8.8, 1 H);
7.02 (d, J¼8.8, 1 H); 3.92 (s, 3 H); 3.27, 3.16 (AB, JAB ¼16.6, 2 H); 1.34 (s, 3 H); 1.10–1.85 (m, 5 H); 1.00
(d, J¼6.7, 6 H). Anal. calc. for C16H22O5 (294.34): C 65.29, H 7.53; found: C 65.01, H 7.17.
Methyl 4-Hydroxy-3-(3-hydroxy-3,7-dimethyloctanoyl)benzoate (4c). Yield 73%. M.p. 60–618. Rf
(AcOEt/hexane 25 :75) 0.49. 1H-NMR (CDCl3): 12.6 (s, 1 H); 8.51 (d, J¼2.2, 1 H); 8.16 (dd, J¼2.2, 8.8,
1 H); 7.04 (d, J¼8.8, 1 H); 3.95 (s, 3 H); 3.08–3.37 (m, 2 H); 1.01–1.71 (m, 10 H); 0.89 (d, J ¼ 7.0, 2 H).
Anal. calc. for C18H26O5 (322.40): C 67.06, H 8.13; found: C 66.88, H 7.98.
Methyl 4-Hydroxy-3-(3-hydroxy-3-methylnonanoyl)benzoate (4d). Yield 79%. Rf (AcOEt/hexane
25 :75) 0.67. 1H-NMR (CDCl3): 12.57 (s, 1 H); 8.49 (d, J¼1.7, 1 H); 8.14 (dd, J¼1.7, 8.7, 1 H); 7.02 (d, J ¼
8.7, 1 H); 3.93 (s, 3 H); 3.28, 3.20 (AB, JAB ¼16.5, 2 H); 1.56–1.75 (m, 2 H); 1.21–1.46 (m, 13 H). Anal.
calc. for C18H26O5 (322.40): C 67.06, H 8.13; found: C 67.90, H 8.43.
Methyl 3-(3-Hydroxy-3,7-dimethyloct-6-enoyl)-4-methoxybenzoate (11). Yield 56%. Rf (AcOEt/
hexane 25 :75) 0.31. 1H-NMR (CDCl3): 8.30 (d, J¼2.2, 1 H); 8.16 (dd, J ¼ 2.2, 8.8, 1 H); 7.00 (d, J ¼ 8.8,
1 H); 5.01–5.12 (m, 1 H); 3.96 (s, 3 H); 3.90 (s, 3 H); 3.17, 3.09 (AB, JAB ¼16.6, 2 H); 2.50–2.70 (m, 8 H);
1.91–2.16 (m, 2 H); 1.30 (s, 3 H). Anal. calc. for C19H26O5 (334.41): C 68.24, H 7.84; found: C 68.88, H
7.25.
General Procedure for the Synthesis of Compounds 5b–5d and 12. The appropriate ester 4 or 11
(0.42 mmol), resp., was dissolved in MeOH (3.5 ml) and then cooled to 08. NaBH4 (0.048 g, 1.26 mmol)
was added portionwise, and the mixture was slowly (without removing ice bath) warmed to r.t. After 2 h
from the addition of NaBH4, MeOH was evaporated in vacuo. The residue was diluted with AcOEt,
followed by addition of sat. aq. NH4Cl soln. The org. layer was separated, dried (anh. Na2SO4), filtered,
and concentrated to afford the products 5 and 12, resp., as thick viscous liquids.
Methyl 3-(1,3-Dihydroxy-3,5-dimethylhexyl)-4-hydroxybenzoate (5b). Yield 75%. Rf (AcOEt/
1
hexane 3 :7) 0.40. H-NMR (CDCl3): 7.91 (dd, J ¼ 1.7, 8.9, 1 H); 7.61 (d, J ¼ 1.7, 1 H); 6.90 (d, J ¼ 8.9,
1 H); 5.27–5.40 (m, 1 H); 3.87 (s, 3 H); 2.10–2.26 (m, 2 H); 1.35 (s, 3 H); 1.20–1.90 (m, 3 H); 0.86 (d, J ¼
6.7, 6 H). Anal. calc. for C16H24O5 (296.36): C 64.84, H 8.16; found: C 65.01, H 7.98.
Methyl 3-(1,3-Dihydroxy-3,7-dimethyloctyl)-4-hydroxybenzoate (5c). Yield 80%. Rf (AcOEt/hexane
3 :7) 0.45. 1H-NMR (CDCl3): 9.69 (s, 1 H); 7.85 (dd, J ¼ 1.9, 8.6, 1 H); 7.63 (d, J ¼ 1.9, 1 H); 6.88 (d, J ¼
8.6, 1 H); 5.72 (br. s, 1 H); 5.61 (br. s, 1 H); 5.29–5.46 (m, 1 H); 3.89 (s, 3 H), 2.12–2.25 (m, 1 H); 1.10–
1.82 (m, 10 H); 0.94 (d, J ¼ 6.7, 6 H). Anal. calc for C18H28O5 (324.41): C 66.64, H 8.70; found: C 66.21, H
8.45.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Methyl 3-(1,3-Dihydroxy-3-methylnonyl)-4-hydroxybenzoate (5d). Yield 82%. Rf (AcOEt/hexane
3 :7) 0.50. 1H-NMR (CDCl3): 9.40 (s, 1 H); 7.83 (dd, J ¼ 1.9, 8.6, 1 H); 7.65 (d, J ¼ 1.9, 1 H); 6.86 (d, J ¼
8.6, 1 H); 5.72 (br. s, 1 H); 5.29–5.39 (m, 1 H); 3.89 (s, 3 H); 2.15–2.23 (m, 2 H); 1.10–1.82 (m, 13 H);
0.94 (t, J ¼ 6.7, 3 H). Anal. calc. for C18H28O5 (324.41): C 66.64, H 8.70; found: C 66.40, H 8.33.
Methyl 3-(1,3-Dihydroxy-3,7-dimethyloct-6-en-1-yl)-4-methoxybenzoate (12). Yield 83%. Rf
1
(AcOEt/hexane 1:1) 0.40. H-NMR (CDCl3): 8.11 (d, J ¼ 2.2, 1 H); 7.97 (dd, J ¼ 2.2, 8.8, 1 H); 6.90
(d, J ¼ 8.8, 1 H); 5.37–5.62 (m, 1 H); 5.05–5.27 (m, 1 H); 3.93 (s, 3 H); 3.91 (s, 3 H); 1.90–2.16 (m, 2 H);
1.20–1.81 (m, 10 H). Anal. calc. for C19H28O5 (336.42): C 67.83, H 8.39; found: C 67.37, H 8.10.
General Procedure for the Synthesis of Compounds 6b–6d and 13. The appropriate ester 5 or 12
(0.21 mmol), resp., was taken in 2 ml of THF/H2O 3 :1, followed by addition of LiOH·H2O (0.35 g,
0.83 mmol). The resulting mixture was heated to reflux for 6 h and then cooled to r.t. THF was
evaporated, and the remaining aq. layer was cooled to 08 and acidified to pH 3 with cold 2n HCl. The
resulting white precipitate was filtered, washed with H2O, and dried to afford acid 6 and 13, resp., as
sticky solids.
3-(1,3-Dihydroxy-3,5-dimethylhexyl)-4-hydroxybenzoic Acid (6b). Yield quant. Rf (CH2Cl2/MeOH
95 :5) 0.45. 1H-NMR (CDCl3): 12.60 (s, 1 H); 7.91 (dd, J ¼ 1.7, 8.9, 1 H); 7.63 (d, J ¼ 1.7, 1 H); 6.93 (d, J ¼
8.9, 1 H); 5.24–5.32 (m, 1 H); 2.19–2.26 (m, 2 H); 1.36 (s, 3 H); 1.20–1.90 (m, 3 H); 0.86 (d, J ¼ 6.7,
6 H). Anal. calc. for C15H22O5 (282.33): C 63.81, H 7.85; found: C 63.39, H 7.54.
3-(1,3-Dihydroxy-3,7-dimethyloctyl)-4-hydroxybenzoic Acid (6c). Yield 96%. Rf (CH2Cl2/MeOH
95 :5) 0.40. 1H-NMR (CDCl3): 9.80 (s, 1 H); 7.90 (dd, J ¼ 1.7, 8.9, 1 H); 7.67 (d, J ¼ 1.7, 1 H); 6.90 (d, J ¼
8.9, 1 H); 5.29–5.46 (m, 1 H); 2.08–2.27 (m, 2 H); 1.05–1.94 (m, 10 H); 1.35 (s, 3 H); 0.86 (d, J ¼ 6.7,
6 H). Anal. calc. for C17H26O5 (310.39): C 65.78, H 8.44; found: C 65.54, H 8.10.
3-(1,3-Dihydroxy-3-methylnonyl)-4-hydroxybenzoic Acid (6d). Yield 67%. Rf (CH2Cl2/MeOH 95 :5)
0.30. 1H-NMR (CDCl3): 9.40 (s, 1 H); 7.83 (dd, J ¼ 1.9, 8.6, 1 H); 7.67 (d, J ¼ 1.9, 1 H); 6.88 (d, J ¼ 8.6,
1 H); 5.27–5.39 (m, 1 H); 2.20–2.27 (m, 2 H); 1.10–1.90 (m, 13 H); 0.87 (t, J ¼ 6.7, 3 H). Anal. calc. for
C17H26O5 (310.39): C 65.78, H 8.44; found: C 65.34, H 8.07.
3-(1,3-Dihydroxy-3,7-dimethyloct-6-en-1-yl)-4-methoxybenzoic Acid (13). Yield 99%. Rf (CH2Cl2/
MeOH 95 :5) 0.40. 1H-NMR (CDCl3): 8.15 (d, J ¼ 1.9, 1 H); 7.93 (dd, J ¼ 1.9, 8.8, 1 H); 6.92 (d, J ¼ 8.8,
1 H); 5.35–5.50 (m, 1 H); 5.04–5.25 (m, 1 H); 3.89 (s, 3 H); 1.98–2.21 (m, 2 H); 1.15–1.87 (m, 10 H).
Anal. calc. for C18H26O5 (322.40): C 67.07, H 8.13; found: C 66.85, H 8.00.
General Procedure for the Synthesis of Compounds 7b–7d and 14. To a stirred soln. of IBX (0.149,
0.53 mmol) in DMSO (1.5 ml), the appropriate compound 6 or 13 (0.018 mmol; in 1 ml DMSO), resp.,
was added dropwise at 08 under Ar. The mixture was warmed to r.t. and stirred for 3 h. After the mixture
was diluted with H2O and filtered through a pad of Celite, the residue was washed with AcOEt. The
resulting filtrate and washings were combined and dried (anh. Na2SO4), filtered, and concentrated. The
crude product was purified by reversed-phase CC (H2O/MeOH 35 :65) to afford compounds 7 or 14,
resp.
4-Hydroxy-3-(3-hydroxy-3,5-dimethylhexanoyl)benzoic Acid (7b). Yield 68%. M.p. 153–1558. Rf
(CH2Cl2/MeOH 9 :1) 0.55. 1H-NMR (CDCl3): 12.66 (s, 1 H); 8.57 (d, J ¼ 1.7, 1 H); 8.21 (dd, J¼1.7, 8.9,
1 H); 7.07 (d, J¼8.9, 1 H); 3.28, 3.15 (AB, J¼16.6, 2 H); 1.36 (s, 3 H); 1.20–1.88 (m, 3 H); 1.03 (d, J ¼ 6.7,
6 H). 13C-NMR (CDCl3): 207.2; 169.9; 166.8; 137.8; 133.5; 119.9; 119.3; 119.1; 72.6; 27.3; 24.5; 24.4 (ꢁ2);
24.2 (ꢁ2). Anal. calc. for C15H20O5 (280.32): C 64.27, H 7.19; found: C 63.89, H 6.90.
4-Hydroxy-3-(3-hydroxy-3,7-dimethyloctanoyl)benzoic Acid (7c). Yield 69%. M.p. 156–1588. Rf
(CH2Cl2/MeOH 95 :5) 0.64. 1H-NMR (CDCl3): 12.66 (s, 1 H); 8.56 (d, J¼1.7, 1 H); 8.19 (dd, J¼1.7, 8.9,
1 H); 7.05 (d, J¼1.7, 1 H); 3.27, 3.18 (AB, J¼16.6, 2 H); 1.15–1.70 (m, 7 H); 1.35 (s, 3 H); 0.87 (d, J¼6.7,
6 H). Anal. calc. for C17H24O5 (308.37): C 66.21, H 7.84; found: C 65.98, H 7.35.
4-Hydroxy-3-(3-hydroxy-3-methylnonanoyl)benzoic Acid (7d). Yield 70%. M.p. 118–1208. Rf
1
(CH2Cl2/MeOH 9 :1) 0.51. H-NMR (CDCl3): 12.67 (s, 1 H); 8.58 (d, J¼1.7, 1 H); 8.21 (dd, J¼1.7, 8.9,
1 H); 7.07 (d, J ¼ 8.9, 1 H); 3.28, 3.19 (AB, J¼16.6, 2 H); 1.58–1.71 (m, 2 H); 1.19–1.46 (m, 11 H).
13C-NMR (CDCl3): 207.2; 170.1; 166.7; 137.8; 133.5; 120.0; 119.3; 119.0; 72.2; 46.8; 42.3; 31.5; 29.5; 26.9;
23.7; 22.4; 13.9. Anal. calc. for C17H24O5 (308.37): C 66.21, H 7.84; found: C 65.87, H 7.47.
3-(3-Hydroxy-3,7-dimethyloct-6-enoyl)-4-methoxybenzoic Acid (14). Yield 70%. M.p. 108–1108. Rf
(CH2Cl2/MeOH 94 :6) 0.5. 1H-NMR (CDCl3): 8.39 (d, J¼2.2, 1 H); 8.24 (dd, J¼2.2, 8.6, 1 H); 7.06 (d, J¼

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
47
8.6, 1 H); 5.04–5.18 (m, 1 H); 4.01 (s, 3 H); 3.22, 3.13 (AB, JAB ¼16.6, 2 H); 1.94–2.16 (m, 2 H); 160–1.70
(m, 8 H); 1.31 (s, 3 H). 13C-NMR (CDCl3): 203.2; 170.0; 162.0; 135.5; 132.4; 131.4; 128.7; 124.0; 121.7;
111.3; 72.1, 55.8; 52.0; 41.8; 26.6; 25.4; 22.5; 17.4. Anal. calc for C18H24O5 (320.38): C 67.48, H 7.55; found:
C 67.18, H 7.25.
Antifungal Activity. Cladosporium cladosporioides (F 167) was used for antifungal assay. For the
antifungal assay, 10.0 ml of solns., corresponding to 50.0, 40.0, 30.0, 20.0, 10.0, 5.0, 1.0, 0.5, and 0.1 mg, were
applied to precoated SiO2 TLC plates. The chromatograms were sprayed with a spore suspension of C.
cladosporioides in maltose and salt soln., and incubated for 72 h in darkness in a moistened chamber at
258, following the procedure in [4–6]. Fungal growth inhibition appeared as clear zones against a dark
background, indicating the minimum amount of compounds required for it (Table). Commercial
fungicides prochloraz and cyproconazole were used as controls.
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Received August 30, 2011