Rational design of antimicrobial agents: Antifungal activity of alk(en)yl dihydroxybenzoates and dihydroxyphenyl alkanoates

Article abstract of DOI:10.1016/j.bmcl.2003.08.057

A homologous series (C₃-C₁₄) of each alkyl 3,4- and 3,5-dihydroxybenzoates, and 3,4- and 3,5-dihydroxyphenyl alkanoates exhibit similar antifungal activity against Saccharomyces cerevisiae. Their nonyl derivatives exhibit the most potent antifungal activity against this yeast with the minimum fungicidal concentration (MFC) in the range between 12.5 and 50 μg/mL. In addition, various 3,4-dihydroxybenzoates, possessing different side chains, namely unsaturated, branched and alicyclic were synthesized and their activity was compared.

Full text of DOI:10.1016/j.bmcl.2003.08.057

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3993–3996
Rational Design of Antimicrobial Agents: Antifungal Activity of
Alk(en)yl Dihydroxybenzoates and Dihydroxyphenyl Alkanoates
Ken-ichi Nihei, Atsuko Nihei and Isao Kubo*
Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA
Received 18 June 2003; revised 15 August 2003; accepted 27 August 2003
Abstract—A homologous series (C3–C14) of each alkyl 3,4- and 3,5-dihydroxybenzoates, and 3,4- and 3,5-dihydroxyphenyl
alkanoates exhibit similar antifungal activity against Saccharomyces cerevisiae. Their nonyl derivatives exhibit the most potent
antifungal activity against this yeast with the minimum fungicidal concentration (MFC) in the range between 12.5 and 50 mg/mL. In
addition, various 3,4-dihydroxybenzoates, possessingdifferent side chains, namely unsaturated, branched and alicyclic were syn-
thesized and their activity was compared.
# 2003 Elsevier Ltd. All rights reserved.
In our previous reports on structure–antifungal activity
relationship (SAR) studies with a series of aliphatic
alcohols using Saccharomyces cerevisiae ATCC 7754 as
a model, their maximum antifungal activity was descri-
bed to be dependent on the hydrophobic alkyl (tail)
chain length from the hydrophilic hydroxyl group
(head).1 Biophysical processes appears to be a major
contributor to the antimicrobial activity of amphipathic
alkanols. Antifungal agents that primarily act as sur-
factants may target the extracytoplasmic region and
thus do not need to enter the cell, they avoid most cel-
lular pump-based resistance mechanisms. Structurally
simple alkanols were a superior example to understand
the role of the hydrophobic alkyl portion. However,
alkanols may not be an ideal model to proceed with
further SAR study since their derivatization is limited.
This restriction limits the synthesis of many diverse
related molecules for comparison that is essential for
SAR study. On the basis of the data obtained, the
hydrophilic hydroxyl group can be replaced by any
hydrophilic groups as long as the ‘head and tail’ struc-
ture is balanced. This prompted us to search for novel
antifungal agents through synthetic optimization.
selected phenolic acids themselves was tested against S.
cerevisiae first. All possessed little or no activity against
this yeast. For example, 3,4,5-trihydroxybenzoic acid
(gallic acid) (1) did not exhibit any fungicidal activity up
to 3200 mg/mL. However, this phenolic acid was first
selected as the hydrophilic (head) part because gallic
acid is a common natural product isolated from many
edible plants and more importantly, its three esters—
propyl, octyl, and dodecyl (lauryl)— are currently per-
mitted for use as antioxidant additives in food.2 Fur-
thermore, synthesis of a series of the esters and their
related analogues is essential for SAR study and this
can be easily carried out. It should be noted that various
additional biological activities, which are needed to pro-
tect foods can be introduced by selectingappropriate
head portions.
Amongthe alkyl 3,4,5-trihydroxybenzoates tested,
nonyl 3,4,5-trihydroxybenzoate (C9) (2) was found to be
the most effective against S. cerevisiae with the mini-
mum fungicidal concentration (MFC) of 12.5 mg/mL
(42 mM), followed by octyl 3,4,5-trihydroxybenzoate (3)
with an MFC of 25 mg/mL (89 mM).3 Interestingly,
decyl 3,4,5-trihydroxybenzoate (C10) (4) did not show
any activity.3 It is worth notingthat undecyl (C 11) (5)
and dodecyl (C12) (6) 3,4,5-trihydroxybenzoates did not
exhibit any antifungal activity against S. cerevisiae, but
still showed antibacterial activity against Gram-positive
bacteria.4 The maximum activity of the carbon chain
lengths in alkyl 3,4,5-trihydroxybenzoates differed
between the microorganisms tested, similar to those
In order to have more diverse structural analogues,
various phenolic acids were selected as alternative
hydrophilic head portions. The antifungal activity of the
*Correspondingauthor. Tel.: +1-510-643-6303; fax: +1-510-643-
0215; e-mail: ikubo@uclink.berkeley.edu
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.057

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K. Nihei et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3993–3996
found for alkanols.5 The antifungal activity of alkyl
3,4,5-trihydroxybenzoates is due primarily to their abil-
ity to act as nonionic surface-active agents (surfactants)
and the alkyl chain length is largely related to the
activity.3,6,7
decanoate were carried out by one step esterification
utilizing N,N0-dicyclohexylcarbodiimide (DCC) as an
3
activatingreaegnt.
However, this method was not
appropriate for syntheses of 7–13, 15, and 16 because of
low yield and hard for eliminatingremainingalcohol.
The correspondingbenzyloxybenzoic acids were
obtained by the methods previously reported.8ꢀ14 The
final product alkyl hydroxybenzoates, were obtained in
high yield by two step procedures, Mitsunobu reaction
as a key step followed by hydrogenation for the removal
Subsequently, nonyl 2-hydroxybenzoate (7), nonyl 3-
hydroxybenzoate (8), nonyl 4-hydroxybenzoate (9),
nonyl 2,3-dihydroxybenzoate (10), nonyl 2,4-
dihydroxybenzoate (11), nonyl 2,5-dihydroxybenzoate
(12), nonyl 3,5-dihydroxybenzoate (13), nonyl 3,4-dihy-
droxybenzoate (protocatechuate) (14), nonyl 3-hydroxy-4-
methoxybenzoate (15), nonyl 4-hydroxy-3-methoxy-
benzoate (16), 3,4-dihydroxyphenyl decanoate (17), and
3,5-dihydroxyphenyl decanoate (18) were synthesized (Fig.
1). The synthesis of nonyl 3,4-dihydroxybenzoate, 3,4-
dihydroxyphenyl decanoate, and 3,5-dihydroxyphenyl
15
of protectinggroup.
The synthesized nonyl hydroxybenzoates (7–16) were
tested for their antifungal activity against S. cerevi-
siae.16,17 Amongthem, nonyl 3,5-dihydroxybenzoate
and nonyl 3,4-dihydroxybenzoate were the only active
compounds each with MFC of 12.5 mg/mL, respec-
tively.18 In contrast, neither monohydroxybenzoates (7–
9) nor salicylic acid derivatives (10–12) showed any
antifungal activity. It appears therefore that two
hydroxyl groups are essential to elicit the activity as the
hydrophilic ‘head’ portion. This can be supported by
the observation that neither nonyl 3-hydroxy-4-meth-
oxybenzoate nor nonyl 4-hydroxy-3-methoxybenzoate
showed any fungicidal activity. In the case of nonyl 2,3-,
2,4-, and 2,5-dihydroxybenzoate, the salicylate moiety
of ‘a’ is in a sterically crowded environment and forms
an intramolecular hydrogen bond. This may suggests
that the moiety of a is not able to bind the membrane
surface and hence cannot act as surfactants. A small
change in chemical structures, especially in the head
portion, was noted to affect biological activity to a large
extent.
The ‘hydrolyzable’ ester group was selected in order to
avoid undesired side effects; particularly endocrine dis-
ruptingactivity of environmentally persistent estrogen
mimics19 such as alkylphenolic compounds.20 The ester
group is not directly related to the activity since 3,4- and
3,5-dihydroxyphenyl decanoate exhibited the compar-
able fungicidal activity against S. cerevisiae with that of
nonyl 3,4-dihydroxybenzoate. The possibility that S.
cerevisiae cells do secrete nonspecific extracellular
esterases that hydrolyzes nonyl 3,4-dihydroxybenzoate
to 3,4-dihydroxybenzoic acid (19) and 1-nonanol (20),
the antifungal activity observed could be expected from
the hydrolysates. However, the MIC and MFC of 1-
nonanol against S. cerevisiae were 100 and 200 mg/mL,
respectively. 3,4-Dihydroxybenzoic acid (19) did not
exhibit any antifungal activity against S. cerevisiae up to
3200 mg/mL.
The same series of alkyl 3,4-dihydroxybenzoates was
synthesized and tested for their antifungal activity
against the same S. cerevisiae strain for comparison.
The results are listed in Table 1. The potency of alkyl
3,4-dihydroxybenzoates is noted to be nearly compar-
able with those of the correspondingalkyl 3,4,5-trihy-
droxybenzoates,3 indicatingthat the additional
hydroxyl group is not essential. The range of the anti-
fungal activity of alkyl 3,4-dihydroxybenzoates tested
against S. cerevisiae is between 12.5 and 400 mg/mL.
The maximum potency of both the growth inhibitory
Figure 1. Structures of alkyl hydroxybenzoates.

K. Nihei et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3993–3996
3995
Table 1. Antifungal activity of alkyl 3,4-dihydroxybenzoate against
S. cerevisiae ATCC 7754
Table 2. Antifungal activity of bulky alkyl 3,4-dihydroxybenzoates
against S. cerevisiae ATCC 7754
Compd tested
mg/mL
log P
Comp tested
mg/mL
log P
MIC (MFC)
MIC (MFC)
C3
C4
C5
C6
C7
C8
C9
C10
>400 (>400)
200 (400)
100 (100)
1.90
2.32
2.73
3.15
3.57
3.99
4.40
4.82
5.24
—
trans-2-Nonene-1-yl (22)
cis-2-Nonene-1-yl (23)
2-Nonanyl (24)
6.25 (6.25)
6.25 (6.25)
25 (25)
12.5 (12.5)
50 (50)
12.5 (12.5)
12.5 (25)
12.5 (12.5)
50 (50)
4.22
4.22
4.30
4.37
3.05
3.84
3.84
4.10
3.78
3.62
25 (25)
3-Nonanyl (25)
12.5 (12.5)
6.25 (12.5)
6.25 (12.5)
>400 (>400)
>400 (>400)
6.25 (50)
Cyclohexylmethyl (26)
Geranyl (27)
Neryl (28)
Menthyl (29)
Bornyl (30)
Decahydro-2-naphtyl (31)
C11
Miconazole
25 (25)
Numbers in italic type in parentheses are MFC.
Log P values were calculated by the method described.21
Numbers in italic type in parentheses are MFC.
(MIC) and the fungicidal effect (MFC) was found in
octyl (C8) (21) and nonyl (C9) (14) 3,4-dihydroxy-
benzoates each at 6.25 and 12.5 mg/mL, respectively.
The differences between the MIC and MFC values are
not more than 2-fold, suggesting that their activity is
fungicidal.
As longas the head and tail structure is balanced, the
hydrophobic portion seems to be flexible. In the surfac-
tant concept, the solubility of molecules in the water
based medium should be an important concern for syn-
thetic optimization. The introduction of branchingor
unsaturation into the hydrophobic group is known to
increase the solubility of the surfactant in water.22 If the
hydrophobic portion of the molecule enters into the
membrane lipid bilayer and creates disorder in the fluid
bilayer, increasingthe volume of the hydrophobic por-
tion through synthetic modification may enhance the
activity. This phenomenon can be expressed by log P
values. Since decyl 3,4,5-trihydroxybenzoate (4) did not
exhibit any antifungal activity against S. cerevisiae, the
target molecule’s log P value should be as close to 4.5
but not largely exceed it. The hydrophobic portion is
not a major contributor but obviously associated with
the activity to a large extent. The more bulky alkyl
groups may form large size pores and as a result, may
increase the activity. Hence, various alcohols were
selected and the log P values of their 3,4-dihydroxy-
benzoates were calculated. The values calculated are
listed in Table 1 and most are around between 3.5 and
4.5. The 3,4-dihydroxybenzoates in the range of this log
P values possess the optimum balance between hydro-
philicity and lipophilicity to act as surfactants.
Figure 2. Structures of bulky alkyl 3,4-dihydroxybenzoates.
Table 3. Antifungal activity of 1,2,4-benzenetriol alkanoates against
S. cerevisiae ATCC 7754
Similarly bulkier alkyl 3,4-dihydroxybenzoates were
synthesized in the same manner.3 The synthesized alkyl
3,4-dihydroxybenzoates; trans-2-nonene-1-yl (22), cis-2-
nonene-1-yl (23), 2-nonanyl (24), 3-nonanyl (25), cyclo-
hexylmethyl (26), geranyl (27), neryl (28), menthyl (29),
bornyl (30), and decahydro-2-naphtyl (31), were assayed
against the same S. cerevisiae strain for comparison
(Fig. 2). The results are listed in Table 2. As expected,
all the alkyl 3,4-dihydroxybenzoates tested, regardless
of their alkyl shape exhibited the activity. The MFC to
MIC ratios are no greater than 2-fold, indicating that
Comps tested
mg/mL
log P
MIC (MFC)
C7
C8
C9
C10
C11
C12
C13
200 (200)
50 (100)
25 (25)
12.5 (25)
25 (25)
3.16
3.58
4.00
4.42
4.83
5.25
5.67
>400 (>400)
>400 (>400)
Numbers in italic type in parentheses are MFC.

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K. Nihei et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3993–3996
their activity is fungicidal. Both trans- and cis-2-nonene-
1-yl-3,4-dihydroxybenzoates were found to be the most
effective against S. cerevisiae each with an MFC of 6.25
mg/mL (23 mM). As far as alkyl 3,4-dihydroxybenzoates
are compared, the compounds possessingsimilar log P
values exhibit similar MFC values, and 6.25 mg/mL of
both trans- and cis-2-nonene-1-yl-3,4-dihydroxy-
benzoates was the maximum activity obtained through
synthetic optimization. Overall, the molecular shape does
not appear to be a major contributor to the activity.
5. Kubo, I.; Muroi, H.; Himejima, M.; Kubo, A. Bioorg.
Med. Chem. 1995, 3, 873.
6. Fujita, K.; Kubo, I. J. Appl. Microbiol. 2002, 92, 1035.
7. Fujita, K.; Kubo, I. Int. J. Food Microbiol. 2002, 79, 193.
8. Zane, A.; Wender, S. H. J. Org. Chem. 1964, 29, 2078.
9. Deng, J.; Hamada, Y.; Shioiri, T. Synthesis 1998, 627.
10. Teshima, T.; Matsumoto, T.; Wakamiya, T.; Shiba, T.;
Aramaki, Y.; Nakajima, T.; Kawai, N. Tetrahedron 1991, 47,
3305.
11. Haddleton, D. M.; Sahota, H. S.; Taylor, P. C.; Yeates,
S. G. J. Chem. Soc., Perkin Trans. 1 1996, 649.
12. Cohen, S. M.; Raymond, K. N. Inorg. Chem. 2000, 39,
3624.
Subsequently, the same series of 3,4-dihydroxyphenyl
alkanoates was synthesized and tested for their anti-
fungal activity against S. cerevisiae for comparison. The
results are similar to those found for alkyl 3,4-dihy-
droxybenzoates as listed in Table 3. 3,4-Dihydroxy-
phenyl nonanoate (Cg), 3-4-dihydroxyphenyl deconoate
(C10) (17) and undecanoate (C11) (32) were found to be
the most effective against S. cerevisiae each with an
MFC of 25 mg/mL. Furthermore, the same series of 3,5-
dihydroxybenzoates and 3,5-dihydroxyphenyl alkano-
ates were synthesized and examined for their antifungal
activity. The results are similar to those found for alkyl
3,4-dihydroxybenzoates (data not listed). The activity
was found to correlate with the hydrophobic alkyl chain
length. The compounds possessing similar log P values
exhibit similar activity.
13. Hu, H.; Mendoza, J. S.; Lowden, C. T.; Ballas, L. M.;
Janzen, W. P. Bioorg. Med. Chem. 1997, 5, 1873.
14. Baggaley, K. H.; Fears, R.; Hindley, R. M.; Morgan, B.;
Murrell, E.; Thorne, D. E. J. Med. Chem. 1977, 20, 1388.
15. Nonyl 3,5-dihydroxybenzoate was synthesized as follows.
A mixture of 3,5-dibenzyloxybenzoic acid (200 mg, 0.60
mmol),11 1-nonanol (95 mg, 0.66 mmol), and PPh3 (190 mg,
0.72 mmol) in THF (4 mL) was cooled to 0 ꢁC and treated
with diisopropyl azodicarboxylate (146 mg, 0.72 mmol). After
beingstirred for 2 h at room temp, the solvent was removed in
vacuo. The residue was subjected to silica gel chromatography
eluted with 1–8% AcOEt–hexane to give an ester as white
solid, which was used in the next step without further pur-
ification. The ester was hydrogenated over 20% Pd(OH)2 on
carbon (10 mg) in 1% AcOH–AcOEt (4 mL) for 12 h. Fil-
tration through Celite and concentration followed by silica
gel chromatography (15–30% AcOEt–hexane) gave 155 mg
(92%) of the title compound as a white solid. The structure
was established by spectroscopic methods (IR, NMR, and
MS).
Acknowledgements
16. The procedures used for antifungal assay were the same as
previously described.3 The highest concentration of some
esters remain uncertainty because of their solubility in limited
the water-based medium.
17. The test strains, S. cerevisiae ATCC 7754 used for this
study was purchased from American Type Culture Collection
(Manassas, VA, USA).3
The authors are grateful to Dr. K. Fujita for his continu-
ingadvises, and Dr. K. Tsujimoto, Dr. K. Shimizu and
Dr. N. Masuoka for obtainingthe spectroscopic data.
18. Nonyl 3,4-dihydroxybenzoate was also found to inhibit
the growth of Candida albicans ATCC 18804 and Zygo-
saccharomyces ATCC 60483 each with MFC of 50 mg/mL.
19. Soto, A. M.; Justicia, H.; Wray, J. W.; Sonnenschein, C.
Environ. Health Perspect. 1991, 92, 167.
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