Synthesis of chlorogenic acid derivatives with promising antifungal activity

Article abstract of DOI:10.1016/j.bmc.2007.07.038

Derivatives of chlorogenic acid or its analogues were synthesized by coupling protected chlorogenic acid or its analogues with p-octyloxyaniline and selected amino acids. Most of the compounds exhibited significant potency against Cryptococcus neoformans and Candida species with low toxicity to brine shrimps. The 4,5-dihydroxyl groups in the quinic acid moiety were necessary for the activity and introduction of a free amino group increased the inhibitory activity against Aspergillus fumigatus.

Full text of DOI:10.1016/j.bmc.2007.07.038

Bioorganic & Medicinal Chemistry 15 (2007) 6830–6833
Synthesis of chlorogenic acid derivatives with promising
antifungal activity
Chao-Mei Ma,a,c Maureen Kully,b Jehangir K. Khan,b
Masao Hattoric and Mohsen Daneshtalaba,*
aSchool of Pharmacy, Memorial University of Newfoundland, St. John’s, NL, Canada A1C 3V6
bNAEJA Pharmaceutical Co. Ltd, 4290-91 A Street, Edmonton, AB, Canada T6E 5V2
cInstitute of Natural Medicine, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan
Received 22 May 2007; revised 11 July 2007; accepted 13 July 2007
Available online 22 August 2007
Abstract—Derivatives of chlorogenic acid or its analogues were synthesized by coupling protected chlorogenic acid or its analogues
with p-octyloxyaniline and selected amino acids. Most of the compounds exhibited significant potency against Cryptococcus neofor-
mans and Candida species with low toxicity to brine shrimps. The 4,5-dihydroxyl groups in the quinic acid moiety were necessary for
the activity and introduction of a free amino group increased the inhibitory activity against Aspergillus fumigatus.
Ó 2007 Elsevier Ltd. All rights reserved.
Life-threatening fungal infections have increased dra-
matically in recent years in immunocompromised pa-
tients such as those undergoing cancer chemotherapy,
organ transplant, and patients with AIDS. As clinically
used antifungal drugs have drawbacks with respect to
side effects and development of drug resistance, discov-
ery and development of new types of antifungal agents
is of high importance.
for 1,3-b-glucan synthase inhibitory activity of echino-
candin-related analogues.3
To examine this hypothesis, we attempted determining
the stereochemically optimized form as well as molecu-
lar dynamic of echinocandin B using Hyperchem-3TM
software on an Octane-2 Silicon Graphic computer.
After molecular optimization of echinocandin B, the
overlay of a series of structurally related natural and
synthetic analogues of homotyrosine on echinocandin
B was performed. Among the phenolic compounds
tried, chlorogenic acid linking with a long chain was
found to have the most favorable spatial relationship
of the essential groups (the phenolic moiety and the lipo-
philic side chain) with those of echinocandin B (unpub-
lished data). Chlorogenic acid is a natural product
existing widely in many vegetables and plants. Chloro-
genic acid linking with one more caffeoyl group (dica-
ffeoylquinic acids) has been reported to have inhibitory
activity on HIV integrase.4 Methyl ester of chlorogenic
acid from natural source has been shown to inhibit
HIV protease.5 Based on the above information we
designed and synthesized chlorogenic acid analogues
possessing a lipophilic chain, including an amino acid
group, at the position 1. The synthesis of chlorogenic
acid derivatives was carried out as depicted in Schemes
1–3. Chlorogenic acid was treated with dried acetone
in the presence of catalytic amount of concentrated
H2SO4 to afford its acetonide derivative, which was then
condensed with 4-(octyloxy) aniline in the presence of
Some natural products and their semisynthetic deriva-
tives have been known as effective antifungal agents
for decades. Nystatin and amphotericin B have long
been used clinically for the treatment of fungal infec-
tions. Candin class of cyclic peptides have recently been
developed as effective drugs to treat opportunistic and
invasive fungal infections.1 We have previously reported
the synthesis of peptidomimetic analogues of echinocan-
din B.2 In that report, we presumed that a suitable posi-
tion of homotyrosine ring (a phenolic moiety), with
respect to the lipophilic side chain, is the determining
factor for the antifungal activity of echinocandins. This
assumption was in agreement with that suggested by
Zambias et al., with respect to structural requirement
Keywords: Chlorogenic acid; p-Octyloxyaniline; Candida albicans;
Cryptococcus neoformans; Aspergillus fumigatus; H2N-aa-4-(octyloxy)
aniline; Brine shrimp lethality assay; Echinocandin B; 1,3-b-Glucan;
1H NMR; ESI-MS; FAB-MS.
*
Corresponding author. Tel.: +1 709 777 6958; fax: +1 709 777
7044; e-mail: mohsen@mun.ca
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.038

C.-M. Ma et al. / Bioorg. Med. Chem. 15 (2007) 6830–6833
6831
CMC and HOBT to obtain compound 1. Deprotection
of compound 1 under acidic condition yielded com-
pound 2.
Both compounds 1 and 2 showed antifungal activity
(Table 1). To investigate the influence of physicochemi-
cal properties on the antifungal activity, selected amino
acids were introduced to the above structures. For the
synthesis of these compounds, protected amino acid
derivatives (Fmoc-Thr(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH,
Fmoc-Asp(OtBu)-OH or Fmoc-Orn(Boc)-OH) were
condensed with 4-(octyloxy) aniline in the presence of
CMC and HOBT to obtain the amino acid derivatives
with lipophilic chains. These compounds were deprotec-
ted with NHEt2 to obtain compounds with free amino
groups, H2N-aa-4-(octyloxy) aniline (Scheme 2). The
H2N-aa-4-(octyloxy) aniline was reacted with the aceto-
nide of chlorogenic acid to obtain compounds 3–6. Acid
hydrolysis of compounds 3–6 was performed under con-
trolled condition to obtain compounds 7–10. Further
de-protection of 7–10 with 90% TFA afforded com-
pounds 11–14. The acid concentration, hydrolysis time,
and temperature were carefully controlled to obtain
stepwised products and to avoid the hydrolysis of the es-
ter bond between caffeoyl and quinic acid moieties.
Scheme 1. Synthesis of chlorogenic acid derivatives 1 and 2. Reagents
and conditions: (a) dry acetone, concd H2SO4; (b) 4-(octyloxy) aniline,
CMC, HOBT; (c) acetone-H2O, 0.67 N HCl, rt, 1 h.
Scheme 2. Synthesis of H2N-aa-4-(octyloxy) aniline. Reagents and
conditions: (a) CMC, HOBT; (b) 10% NHEt2, DMF, rt, 2 h.
To investigate the influence of the caffeoyl moiety on the
bioactivity, compounds 15–19 were synthesized. Hydro-
genation of compound 10 in EtOH containing Pd/C pro-
duced compound 15. Part of compound 15 was
deprotected with 90% TFA to obtain compound 16.
Quinic acid bisacetonide was synthesized according to
the reported method.6 Condensation of acetylcoumaryl
Table 1. Antifungal activity of chlorogenic acid derivatives
Compound
MICa,b (lg/ml)
% BLc
C. albicans C. neoformans A. fumigatus
1
16
8
2
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
16
100.0
50.0
31.3
6.2
2
2
3
32
32
>64
>64
4
2
4
4
5
2
56.2
12.5
31.1
50.0
37.5
25.0
18.8
12.5
6.3
6
2
7
2
8
2
1
9
2
2
10
11
12
13
14
15
16
17
18
19
2
1
>64
8
4
2
64
4
16
1
37.5
21.1
10.5
0
>64
16
>64
>64
8
4
>64
64
8
>64
>64
4
>64
>64
32
31.6
100
Fluconazole 0.25
0.5
>64
a The MIC value was determined by methods of NCCLS. The final
concentration of antifungal agents was between 0.12 and 64 lg/ml.
MICs were read at 80% inhibition.
Scheme 3. Synthesis of chlorogenic acid derivatives 3–14. Reagents
and conditions: (a) CMC, HOBT; (b) acetone-H2O, 0.67 N HCl, rt,
1 h; (c) 90% TFA, rt, 30 min.
b Tested organisms: Candida albicans ATCC90028, Cryptococcus neo-
formans ATCC32045, and Aspergillus fumigatus ATCC13073.
c BL: (%) brine shrimp lethality at 100 lg/ml.

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C.-M. Ma et al. / Bioorg. Med. Chem. 15 (2007) 6830–6833
chloride with the bisacetonide followed by deprotection
yielded chlorogenic acid analogue A (Scheme 4).
in its structure, which had MIC of 16 lg/ml. It has been
reported that incorporation of an amino group (such as
aminoproline residue) into the ring of echinocadin ana-
logues led to improvement of its antifungal potency.9
Similar effects were observed in the present chlorogenic
acid derivatives. Compound 14 with a free amino group
in its structure showed good activity against all the fungi
tested, including A. fumigatus (MIC of 16 lg/ml). The
MIC of the chlorogenic acid derivatives against C. albi-
cans varied from 2 to >64 lg/ml. All the acetonide com-
pounds (1, 3–6) showed weaker inhibitory activity
against C. albicans than the corresponding compounds
with free hydroxyl groups (2, 7–10), suggesting that
the two hydroxyl groups in the quinic acid part are
essential for the activity.
Compound A was protected as acetonide using the same
condition as depicted in Scheme 1. The acetonide deriv-
ative of A was used as a starting material to synthesize
compounds 17–19 by the same method as described in
Scheme 3. The structures of compounds 15–19 are
shown in Figure 1.
The synthesized compounds were evaluated for
in vitro antifungal activity against Candida albicans
ATCC90028, Cryptococcus neoformans ATCC32045,
and Aspergillus fumigatus ATCC13073 according to
the NCCLS guidlines.7,8 Plates were incubated at
35 °C for 18–24 h for C. albicans, 48 h for A. fumigatus,
and 48–72 h for C. neoformans. The MIC values were
determined as the lowest concentration of the com-
pound that inhibited growth up to 80%. The results
are summarized in Table 1.
To test the general toxicity of these compounds, brine
shrimp lethality assay was carried out according to the
reported method.10 Compounds 1 and 19 showed high
toxicity toward the brine shrimps at 100 lg/ml. Com-
pounds 2, 5, and 8 showed moderate toxicity, while
other compounds exhibited low toxicity.
Overall, chlorogenic acid derivatives (1–14) showed bet-
ter antifungal activity or less toxicity than those of
chlorogenic acid analogues (15–19), suggesting that
structural modification on the caffeoyl group, such as
saturating the double bond (16 vs 14) or reducing the
number of hydroxyl groups (19 vs 14), did not benefit
the bioactivity profile. Significant antifungal activity
was observed in most of the chlorogenic acid derivatives
(1–14). The MICs on C. neoformans of nearly all these
chlorogenic acid derivatives were as low as 1–4 lg/ml,
except compound 13 with a free carboxylic acid group
Compounds 8 and 12, due to the existence of threonine
in their molecular structures which make them structur-
ally more resemble to the south-eastern component of
echinocandin, were further tested for their inhibitory
activity against two Candida species, including the drug
(azole)-resistant fungus C. krusei, in comparison with
the parent compound, chlorogenic acid. As shown in
Table 2, both 8 and 12 showed much more potent inhib-
itory activity against the fungi but less toxicity on brine
shrimp than chlorogenic acid.
With respect to mechanism(s) of action of this novel class
of chlorogenic acid derivatives, based on their partial
structural similarity with that of echinocandin B, we
anticipated the same mode of action for these com-
pounds as that of echinocandin B analogues, which is
inhibition of 1,3-b-glucan synthase. This enzyme is essen-
tial for the biosynthesis of the glucan component of fun-
gal cell wall. While Candida and Aspergillus species are
sensitive toward candin class of antifungals, due to the
presence of 1,3-b-glucan in their cell wall structure, C.
neoformans does not exhibit sensitivity against this class
of compounds. Indeed, the main glucan component in C.
neoformans cell wall is 1,6-b-glucan with a biosynthetic
Scheme 4. Synthesis of chlorogenic analogue A: Reagents and
conditions: (a) acetylcoumaryl chloride, CH2Cl2, pyridine; (b) 0.8 N
HCl, THF-H2O, rt, 48 h.
Table 2. Comparison of the bioactivity of 8, 12 with chlorogenic acid
Compound
MICa,b (lg/ml)
% BLc
C. albicans
C. krusei
8
12
4
2
2
50.0
12.5
100
4
Chlorogenic acid
Amphotericin B
>64
0.5
>64
1
a The MIC value was determined by methods of NCCLS. The final
concentration of antifungal agents was between 0.12 and 64 lg/ml.
The MIC values are defined as the lowest concentration showing 80%
inhibition of drug-free control growth.
b Tested organisms: Candida albicans ATCC90028, Candida krusei
ATCC6258.
c BL: (%) brine shrimp lethality at 100 lg/ml.
Figure 1. Structures of compounds 15–19.

C.-M. Ma et al. / Bioorg. Med. Chem. 15 (2007) 6830–6833
6833
pathway different from that of 1,3-b-glucan. Candida
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genic acid derivatives may exhibit their antifungal effect
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In conclusion, we synthesized a novel series of lipo-
chlorogenic acid derivatives. Most of the synthesized
compounds exhibited potent antifungal activity against
Cryptococcus and Candida species but lower toxicity to
brine shrimps than the parent chlorogenic acid.
These novel compounds may serve as leads for the
development of less toxic drugs with different structural
feature from those of the currently utilized antifungal
agents. The structure–activity relationships discussed
above can provide useful information for further design
and synthesis of compounds with optimized bioactivity
profile. Further studies to optimize the structural feature
as well as to explore the mechanism of action of this no-
vel class of compounds is planned to start in our group
in near future.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2007.
07.038.