Synthesis, anti-fungal and 1,3-β-d-glucan synthase inhibitory activities of caffeic and quinic acid derivatives

Article abstract of DOI:10.1016/j.bmc.2010.08.022

New derivatives of caffeic acid and quinic acid were synthesized and their anti-fungal and inhibitory activities on fungal 1,3-β-glucan synthase were determined in comparison with those of the corresponding chlorogenic acid derivatives. All the chlorogenic, quinic and caffeic acid derivatives that were coupled with an H₂N-orn-4-(octyloxy) aniline group (1, 1b and 1c) displayed potent activities in both anti-fungal and inhibition of 1,3-glucan synthase assays. Compounds 1 and 1c inhibited the fungal membrane enzyme with the potency comparable to that of a known 1,3-β-d-glucan synthase inhibitor, aculeacin A. The results revealed that the anti-fungal activity of the chlorogenic acid derivative with a free amino group was at least partly due to inhibition of the fungal 1,3-β-glucan synthase. These results suggest that further investigation on caffeic acid derivatives may lead to the discovery of novel anti-fungal agents with drug-like properties.

Full text of DOI:10.1016/j.bmc.2010.08.022

Bioorganic & Medicinal Chemistry 18 (2010) 7009–7014
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, anti-fungal and 1,3-b-D-glucan synthase inhibitory activities
of caffeic and quinic acid derivatives
Chao-Mei Ma a,b, , Takashi Abe c, Tadazumi Komiyama c, Wei Wang b,d, Masao Hattori b,
⇑
⇑
,
Mohsen Daneshtalab e
a College of Life Sciences, Inner Mongolia University, 235 Daxuexilu, Huhhot, Inner Mongolia 010021, China
b Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
c Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 950-2081, Japan
d Department of Chemistry, School of Pharmaceutical Sciences, Kunming Medical University, Kunming, Yunnan 650500, China
e School of Pharmacy, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada A1B 3V6
a r t i c l e i n f o
a b s t r a c t
Article history:
New derivatives of caffeic acid and quinic acid were synthesized and their anti-fungal and inhibitory
activities on fungal 1,3-b-glucan synthase were determined in comparison with those of the correspond-
ing chlorogenic acid derivatives. All the chlorogenic, quinic and caffeic acid derivatives that were coupled
with an H2N-orn-4-(octyloxy) aniline group (1, 1b and 1c) displayed potent activities in both anti-fungal
and inhibition of 1,3-glucan synthase assays. Compounds 1 and 1c inhibited the fungal membrane
Received 4 June 2010
Revised 8 August 2010
Accepted 10 August 2010
Available online 13 August 2010
enzyme with the potency comparable to that of a known 1,3-b-D-glucan synthase inhibitor, aculeacin
Keywords:
A. The results revealed that the anti-fungal activity of the chlorogenic acid derivative with a free amino
group was at least partly due to inhibition of the fungal 1,3-b-glucan synthase. These results suggest that
further investigation on caffeic acid derivatives may lead to the discovery of novel anti-fungal agents with
drug-like properties.
Caffeic acid derivative
Quinic acid derivative
Chlorogenic acid derivative
Anti-fungal
Ó 2010 Elsevier Ltd. All rights reserved.
Fungal 1,3-b-glucan synthase
1. Introduction
equally important to search for anti-fungal agents that are more
stable in vivo. Caffeic and quinic acid derivatives with no ester
The rapid increase of life-threatening fungal infections along
with the emergence of drug-resistant fungal strains demands the
development of novel anti-fungal agents with new structural fea-
bond in their structures are expected to be more stable in vivo than
the parent chlorogenic acid derivatives. The present research was
carried out to synthesize new caffeic and quinic acid derivatives
tures or new mechanisms of action. 1,3-b-
D-Glucan synthase is
and to evaluate their 1,3-b-D-glucan synthase inhibitory and anti-
an essential enzyme for the synthesis of 1,3-b-glucan component
of fungal cell wall. This enzyme is unique to fungi and is not found
in mammalian cells, thus has been viewed as a promising target for
the development of low toxic anti-fungal agents.1,2 Some chloro-
genic acid derivatives containing lipophilic chains have been re-
ported to have potent anti-fungal activity.3 These compounds
were initially designed to mimic the pharmacophores of a well
fungal activities.
2. Results and discussion
2.1. Synthesis of quinic acid derivatives
Compounds (1–4 and 1p) (Fig. 1) were synthesized using the
known inhibitor of fungal 1,3-b-D-glucan synthase, echinocadin.
previously described procedure with chlorogenic acid as starting
Our recent research showed that though chlorogenic acid deriva-
tives could be absorbed as an intact molecule in vivo, the ester
bond easily broke in vivo to release quinic acid derivatives into
the blood. This absorption and metabolism pattern agreed with
that reported for chlorogenic acid.4 Thus, it is important to investi-
gate if the quinic acid derivatives are active as well. It will be
material.3 Aculeacin, a known 1,3-b-
D-glucan synthase inhibitor,
is used as a positive control in this research.
Alkaline hydrolysis of 2–4 and 1p yielded the corresponding
quinic acid derivatives 2b–4b and the protected quinic acid deriv-
ative 1bp which was de-protected with 90% TFA to yield 1b
(Scheme 1).
The caffeic acid derivatives (1c–4c) were synthesized by allow-
ing 3,4-diacetoxycinnamoyl chloride to react with 4-(octyloxy)
aniline or H2N-aa-4-(octyloxy) aniline followed by de-protection
(Scheme 2). The aa represents an amino acid with the functional
⇑
Corresponding authors. Tel.: +86 15648112473 (C.-M.M.); tel.: +81 76 4347630
(M.H.).
E-mail addresses: cmma@imu.edu.cn (C.-M. Ma), saibo421@inm.u-toyama.ac.jp
(M. Hattori).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.022

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C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 7009–7014
The bioassay results of the synthesized caffeic, quinic and chlor-
ogenic acid derivatives are listed in Table 1. The inhibitory activi-
ties on 1,3-b- -glucan synthase were shown as IC50 representing
D
the concentrations that inhibited 50% of the enzyme activity. The
anti-fungal activities were expressed as MIC. All the chlorogenic
and caffeic acid derivatives showed anti-fungal activity. Of the
three types of compounds, those with a free amino group in their
structures showed stronger activities than those without a free
amino group (1 vs 2–4, 1b vs 2b–4b and 1c vs 2c–4c). The strong
inhibition on 1,3-b-glucan synthase of 1 revealed that the anti-
fungal activity of the chlorogenic acid derivative with a free amino
group was at least partly due to its inhibition on the fungal
1,3-b-glucan synthase. The 1,3-b-glucan synthase inhibitory activ-
ity of chlorogenic acid derivative (1) was similar to that of caffeic
acid derivative (1c) but stronger than that of the quinic acid deriv-
ative (1b). The inhibitory potencies of compounds 1 and 1c on the
fungal membrane 1,3-glucan synthase were similar to that of a
known 1,3-b-D-glucan synthase inhibitor, aculeacin A tested in
the same experiment. The activities demonstrated by the quinic
acid derivative 1b suggested that the chlorogenic acid derivatives
may also display in vivo anti-fungal activity since they release
the corresponding quinic acid derivatives into blood as the major
metabolites according to our recent research results and that re-
ported for chlorogenic acid.4 In addition, the similarity of the 1,3-
Figure 1. Structures of chlorogenic acid derivatives (1–4) and aculeacin A.
b-D-glucan synthase inhibitory and cell-based anti-fungal activities
of the caffeic acid derivative (1c) with that of chlorogenic acid
derivative (1) suggest that further structural modification of caffeic
acid derivatives may lead to the discovery of novel small molecule
anti-fungal agents.
group on its side chain being protected. The H2N-aa-4-(octyloxy)
anilines were synthesized using the procedure described
previously.3
3. Conclusion
2.2. Assessment of anti-fungal and 1,3-b-D-glucan synthase
inhibitory activities
The present study provided synthetic methods for new caffeic
and quinic acid derivatives linking with an amino acid residue
and a lipophilic chain. Quinic acid amides can also be synthesized
using quinic acid as starting material.8,9 Due to the lower cost of
quinic acid than that of chlorogenic acid, the methods described
in the literatures are more economic. When chlorogenic acid is
available as a starting material, the quinic acid derivatives can be
produced with a shorter synthetic route. The starting material,
chlorogenic acid, can be synthesized efficiently from quinic acid.10
An alternative synthetic method for cafffeic acid amides is to use
free caffeic acid to react with amines using benzotriazol-1-yloxy-
The anti-fungal activity on Candida albicans NBRC 1594 was
evaluated according to the NCCLS method.5 The 1,3-b-
-glucan
synthase assay was carried out mainly in two steps. The first step
was to synthesize the glucan in the presence and absence of the
testing compounds by incubating a mixture of 50 mM Tris–HCl
(pH 7.5), 0.5 mM EDTA, 25 mM KF, 20
5.0 mM UDP-Glc with the membrane-bound 1,3-b-
thase. The second step was to measure the amount of the produced
glucan using a 1,3-b- -glucan-dependent enzyme cascade reaction
and a b-glucan determination kit (BGSTAR).
D
l
M GTP
c
-S, 0.75% BSA,
-glucan syn-
D
D
Scheme 1. Synthesis of quinic acid derivatives.

C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 7009–7014
7011
Scheme 2. Synthesis of caffeic acid derivatives.
ing chlorogenic acid derivatives which were previously reported to
have strong anti-fungal activity in vitro.3 The caffeic and quinic
acid derivatives are expected to be more stable in vivo than the
corresponding chlorogenic acid derivatives since as a metabolic
rule, amides are more stable to esterase hydrolysis than are es-
ters.13 The promising in vitro anti-fungal and enzyme inhibitory
activities of the caffeic acid derivatives, along with their possible
in vivo metabolic stability suggest the potential of these com-
pounds as new leads for the discovery of novel small molecule
anti-fungal agents. Further structural optimization on caffeic acid
derivatives and the investigation of their metabolic and pharmaco-
kinetic profiles are planned to be carried out in our group, shortly.
Table 1
Anti-fungal and 1,3-b-D-glucan synthase inhibitory activities
Compound
IC50
(lg/ml)
MICa
(lg/ml)
IC50
(
lM)
MICa
(lM)
1
2
3
4
10
23
4
8
8
32
8
32
>64
>64
2
32
4
32
16
8
16
>64
0.25
15
41
6
14
12
48
16
81
>129
>125
4
84
8.3
64
27
15
29
>181
0.23
>32
>32
27
>32
>32
>32
11
>32
>32
>32
31
>49
>48
53
>81
>65
>63
22
>84
>66
>64
52
1b
2b
3b
4b
1c
2c
3c
4c
1d
3d
4d
Chl
A
4. Experimental
4.1. Materials
20
32
>32
9
37
58
>90
8
The b-glucan determination kit (BGSTAR) was purchased from
Wako, Osaka, Japan. The chemical reagents were purchased from
Sigma-Aldrich, Inc. Column chromatography was carried out on
Chl: chlorogenic acid.
A: aculeacin A.
a
The MIC values were determined by NCCLS method using Candida albicans
Wakogel 50C18 (38–63 lm, Wako Pure Chemical Industries, Ltd).
NBRC 1594.
Preparative HPLC was performed on a Tosoh CCPM-II system
(Tosoh Co., Tokyo) equipped with a UV 8020 detector and a
5C18-AR-II waters HPLC column (20 ꢀ 200 mm). NMR spectra
tris(dimethylamino)phosphonium hexafluorophosphate (BOP) as
the coupling reagent.11,12
Among the compounds described in this paper, all those with an
ornithine residue in their structures exhibited stronger cell-based
anti-fungal and enzyme inhibitory activities. The quinic acid deriv-
atives are considered as the in vivo metabolites of the correspond-
were measured on
a
Varian Unity 500 (1H, 500 MHz; 13C,
125 MHz) or a Varian Gemini 300 (13C, 75 MHz) NMR spectrome-
ter. TMS was used as an internal standard and J values were
reported in Hertz. Electrospray ionization mass (ESI-MS) spectra
were obtained on an Esquire 3000plus spectrometer (Bruker

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C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 7009–7014
Daltonik GmbH, Bremen, Germany). High-resolution-FAB-MS spec-
tra were measured on a Jeol JMS-700 with a resolution of 5000
using m-nitrobenzyl alcohol as the matrix. The purities of synthe-
sized compounds were verified with 1H NMR, 13C NMR and HPLC.
115.4 (C-30,50), 123.3 (C-20,60), 131.6 (C-10), 157.5 (C-40), 175.0
(C-7). ESI-MS (Positive): 396.1 ([M+H]ꢁ, 100%); ESI-MS (Negative):
430.5 ([M+Cl]ꢁ, 100%). HR-FAB-MS [M+H]+ m/z 396.23741 (calcd
for C21H34O6N, requires 396.23861).
4.2. General method for the synthesis of quinic acid derivatives
(1b–4b)
4.2.4. Compound 3b
After synthesis from 3 and purification on a SiO2 column, 3b
was obtained from the EtOAc–MeOH (8:2) eluted part (yield:
94%).1H NMR (CD3OD) d 0.90 (3H, t, J = 6.5 Hz, –(CH2)7CH3), 1.21
(3H, d, J = 6.0 Hz, Thr-CH3), 1.35 (10H, m) and 1.73 (3H, m) and
2.02 (3H, m) (–CH2(CH2)6CH3), H-2 and H-6), 3.42 (1H, br d,
J = 9.0 Hz, H-4), 3.95 (2H, t, J = 6.5 Hz, –CH2(CH2)6CH3), 4.04 (1H,
Compounds 1p and 2–4 were synthesized using the method de-
scribed previously.3 To a methanol solution (6 ml) of compound 1p
or 2–4 (0.05 mmol) was added 4 ml of 4 M NaOH. The mixture was
stirred at rt for 30 min, neutralized with 1 M HCl and condensed
under reduced pressure. The remaining mixture was purified by
chromatography on ODS or on SiO2 to obtain 1bp, and 2b–4b.
m, H-5), 4.17 (1H, br s, H-a), 4.20 (1H, m, H-3), 4.39 (1H, m, H-
b), 6.86 (2H, d, J = 8.5 Hz, H-30,50), 7.41 (2H, d, J = 8.5 Hz, H-20,60).
13C NMR (CD3OD + CDCl3, 125 MHz) d 14.5 (–(CH2)7CH3), 20.3
and 23.7 and 27.2 and 30.4 and 30.5 and 33.0 (–CH2(CH2)6CH3
4.2.1. Compound 1bp
After being synthesized from 1p and purified on an ODS col-
umn, 1bp was obtained from the MeOH–H2O (8:2) eluted part
(yield: 91%).1H NMR (CDCl3) d 0.87 (t, J = 6.5 Hz, 3H, –(CH2)7CH3),
1.28 (19H, m) and 1.49 (2H, m) and 1.70 (3H, m) and 1.85–2.08
and Thr-CH3), 38.8 (C-2), 42.6 (C-6), 59.1 (C-a), 67.3 (C-b, 68.7
(C-5), 69.2 (–OCH2(CH2)6CH3), 72.3 (C-3), 77.0 (C-4), 78.1 (C-1),
115.4 (C-30,50), 123.3 (C-20,60), 131.6 (C-10), 157.5 (C-40), 171.6
(–PhNHCO–), 176.0 (C-7). ESI-MS (Positive): 497.2 ([M+H]ꢁ,
100%); ESI-MS (Negative): 531.5 ([M+Cl]ꢁ, 100%). HR-FAB-MS
[M+H]+ m/z 497.28642 (calcd for C25H41O8N2, requires 497.28628).
(5H, m) (–CH2(CH2)6CH3 and Boc and H2 and H6 and H-
c and
H-b), 2.85 (2H, m, H-d), 3.51 (1H, br s, H-4), 3.84 (2H, t,
J = 6.5 Hz, –CH2(CH2)6CH3), 4.10 (1H, m, H-5), 4.19 (1H, m, H-3),
4.88 (1H, br s, H-
a
), 6.73 (2H, d, J = 8.5 Hz, H-30,50), 7.37 (2H, d,
4.2.5. Compound 4b
J = 8.5 Hz, H-20,60) and some signals for the OH and NH groups.
13C NMR (CDCl3, 75 MHz) d 14.2 (–(CH2)7CH3), 22.7 and 26.1 and
28.5 and 29.3 and 29.4 and 29.5 and 29.8 and 30.2 and 31.9 (–C
After synthesis from 4 and purification on an ODS column, 4b
was obtained from the MeOH–H2O (8:2) eluted part (yield: 93%).
1H NMR (CD3OD) d 0.91 (t, J = 6.5 Hz, 3H, –(CH2)7CH3), 1.35 (8H,
m) and 1.46 (2H, m) and 1.75 (2H, m) (–CH2(CH2)6CH3), 1.90 (1H,
dd, J = 11.0, 13.5 Hz, H-6a), 2.00 (3H, m, H-6b and H-2), 2.83 (2H,
m, H-b (–CH2COOH)), 3.38 (1H, dd, J = 3.5, 9.0 Hz, H-4), 3.95 (2H,
t, J = 6.5 Hz, –CH2(CH2)6CH3), 4.01 (1H, m, H-5), 4.14 (m, 1H, H-
H2(CH2)6CH3 and Boc-CH3 and C-
c), 37.3 (C-2), 39.3 (C-b), 41.4
(C-6), 52.6 (C-
a
), 67.1 (C-5), 68.2 (–OCH2(CH2)6CH3), 71.0 (C-3),
75.8 (C-4), 79.3 (C-1), 114.5 (C-30,50), 121.9 (C-20,60), 130.5 (C-10),
155.9 (Boc-C), 156.7 (C-40), 170.2 (–PhNHCO–), 175.1 (C-7). ESI-
MS (Negative): 608.4 ([MꢁH]ꢁ, 10%).
3), 4.78 (1H, t, J = 6.5 Hz, H-a
), 6.86 (2H, d, J = 8.5 Hz, H-30,50),
Compound 1bp was treated with 90% TFA at rt for 30 min. The
mixture was concentrated to dryness followed by the addition of
2 ml of H2O. The pH of the mixture was then adjusted to 10 by
NH4OH, followed by extraction with EtOAc. The EtOAc layer was
concentrated to dryness to obtain 1b.
7.41 (2H, d, J = 8.5 Hz, H-20,60). 13C NMR (CD3OD, 75 MHz) d 14.5
(–(CH2)7CH3), 23.8 and 27.2 and 30.5 and 33.0 (–CH2(CH2)6CH3),
38.0 (C-b), 38.6 (C-2), 42.4 (C-6), 51.8 (C-a), 68.1 (C-5), 69.2
(–OCH2(CH2)6CH3), 72.1 (C-3), 77.0 (C-4), 78.0 (C-1), 115.5
(C-30,50), 123.2 (C-20,60), 132.0 (C-10), 157.4 (C-40), 170.7
(–PhNHCO–), 174.4 (C-7), 177.0 (COOH). ESI-MS (Positive): 511.2
([M+H]ꢁ, 100%); ESI-MS (Negative): 545.5 ([M+Cl]ꢁ, 100%). HR-
FAB-MS [M+H]+ m/z 511.26470 (calcd for C25H39O9N2, requires
511.26553).
4.2.2. Compound 1b
Yield: 45%, 1H NMR (CD3OD) d 0.93 (t, J = 6.5 Hz, 3H, –(CH2)7CH3),
1.36 (9H, m) and 1.47 (2H, m) and 1.55 (2H, m) and 1.76 (3H, m) and
1.99 (4H, m) (–CH2(CH2)6CH3 and H2 and H6 and H-c and H-b), 2.82
(2H, m, H-d), 3.41 (1H, dd, J = 3.5, 9.0 Hz, H-4), 3.96 (2H, t, J = 6.5 Hz,
4.3. General method for the synthesis of protected caffeic acid
derivatives (1dp, 2p, 3dp and 4dp)
–CH2(CH2)6CH3), 4.04 (1H, m, H-5), 4.17 (1H, m, H-3), 4.51 (1H, dd,
J = 5.5, 8.0 Hz, H-a
), 6.88 (2H, d, J = 8.5 Hz, H-30,50), 7.45 (2H, d,
J = 8.5 Hz, H-20,60). 13C NMR (CD3OD, 75 MHz) d 14.5 (–(CH2)7CH3),
To a solution of 1a–4a (1.5 mmol) and DMAP (68 mg) in CH2Cl2
(10 ml), pyridine (4 ml) and 3,4-diacetoxycinnamoyl chloride
(3.5 mmol) were added at room temperature. The reaction mixture
was stirred overnight and then acidified with 1 M HCl to pH 3. The
aqueous phase was re-extracted with CH2Cl2. The combined organ-
ic extracts were concentrated and purified by SiO2 column chroma-
tography eluted with hexane–EtOAc to yield the products.
23.7 and 27.2 and 27.8 and 30.4 and 30.5 and 31.2 and 33.0
(–CH2(CH2)6CH3 and C-
c), 38.6 (C-2), 41.8 (C-b, C-d), 42.6 (C-6),
54.3 (C- ), 68.1 (C-5), 69.2 (–OCH2(CH2)6CH3), 72.1 (C-3), 76.9
a
(C-4), 78.0 (C-1), 115.5 (C-30,50), 123.0 (C-20,60), 131.9 (C-10), 157.4
(C-40), 171.4 (–PhNHCO–), 176.9 (C-7). ESI-MS (Positive): 510.3
([M+H]ꢁ, 100%); ESI-MS (Negative): 544.6 ([M+Cl]ꢁ, 100%). HR-
FAB-MS [M+H]+ m/z 510.31879 (calcd for C26H44O7N3, requires
510.31792).
4.3.1. Compound 1dp
After synthesis from 1a and purification on SiO2 column, 1dp
was eluted out with hexane–EtOAc (2:8) (yield: 88%). 1H NMR
(CDCl3) d 0.88 (3H, t, J = 6.5 Hz, –(CH2)7CH3), 1.25 (8H, m) and
1.64–1.84 (6H, m) and 2.32 (2H, m) (–CH2(CH2)6CH3 and H-b and
4.2.3. Compound 2b
After synthesis from 2 and purification on a SiO2 column, 2b
was obtained from the EtOAc eluted part (yield: 95%). 1H NMR
(CD3OD) d 0.91 (3H, t, J = 6.5 Hz, –(CH2)7CH3), 1.35 (8H, m) and
1.46 (2H, m) and 1.75 (2H, m) (–CH2(CH2)6CH3), 2.05 (4H, m, H-2
and H-6), 3.42 (1H, dd, J = 3.5, 9.0 Hz, H-4), 3.95 (2H, t, J = 6.5 Hz,
–CH2(CH2)6CH3), 4.04 (1H, m, H-5), 4.17 (1H, m, H-3), 6.87 (2H,
d, J = 8.5 Hz, H-30,50), 7.45 (2H, d, J = 8.5 Hz, H-20,60). 13C NMR
(CD3OD, 75 MHz) d 14.5 (–(CH2)7CH3), 23.8 and 27.2 and 30.4
and 30.5 and 33.0 (–CH2(CH2)6CH3), 38.8 (C-2), 42.6 (C-6), 68.1
(C-5), 69.2 (–OCH2(CH2)6CH3), 72.3 (C-3), 77.0 (C-4), 78.1 (C-1),
H-
(2 ꢀ –COCH3), 3.13 (1H, m) and (3.35 (1H, m) (H-d), 3.87 (2H, t,
J = 6.5 Hz, –CH2(CH2)6CH3), 4.99 (1H, br s, H- ), 6.48 (1H, d,
c), 1.37 (9H, m, H-Boc), 2.27 (3H, s) and 2.28 (3H, s)
a
J = 15.5 Hz, H-8), 6.77 (2H, J = 8.5 Hz, H-30,50), 7.10 (d, J = 8.5,
H-5), 7.26 (1H, d, J = 2.5 Hz, H-2), 7.40 (3H, m, H-20,60,6), 7.50
(1H, d, J = 15.5 Hz, H-7), and NH signal. 13C NMR (CDCl3,
125 MHz) d 14.2 (–(CH2)7CH3), 20.6 and 20.7 and 22.7 and
26.1 and 26.7 and 28.5 and 29.3 and 29.4 and 30.0 and 31.8

C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 7009–7014
7013
(–CH2(CH2)6CH3 and Boc-CH3 and C-b and C-
c
and 2 ꢀ –COCH3),
ture was partitioned with CHCl3 and H2O. The CHCl3 layer was con-
centrated to dryness to obtain the products.
39.4 (C-d), 53.0 (C- ), 68.2 (–OCH2(CH2)6CH3), 79.3 (Boc-C), 114.5
a
(C-30,50), 121.4 (C-6), 121.8 (C-20,60), 122.4 (C-8), 123.7 (C-2),
126.0 (C-5), 130.5 (C-1), 133.4 (C-10), 139.5 (C-7), 142.1 (C-4),
142.8 (C-3), 155.8 (C-40), 156.8 (Boc-CO), 165.8 (C-9), 167.8 and
167.9 (2 ꢀ –COCH), 170.1 (Orn-CO).
4.4.1. Compound 1d
Yield: 93%, 1H NMR (CDCl3) d 0.88 (3H, t, J = 6.5 Hz, –(CH2)7CH3),
1.25–1.93 (25 H, m) (–CH2(CH2)6CH3 and H-b and H-
3.09 (1H, m) and 3.35 (1H, m) (H-d), 3.87 (2H, t, J = 6.5 Hz,
–CH2(CH2)6CH3), 4.81 (1H, br s, H- ), 6.20 (1H, d, J = 15.5 Hz, H-
c and H-Boc),
4.3.2. Compound 2p
a
After synthesis from 2a and purification on a SiO2 column, 2p
was obtained from hexane–EtOAc (6:4) eluted part (yield:
91%).1H NMR (CDCl3) d 0.89 (t, J = 6.5 Hz, 3H, –(CH2)7CH3), 1.30
(8H, m) and 1.44 (2H, m) and 1.77 (2H, m) (–CH2(CH2)6CH3), 2.31
(6H, s, 2 ꢀ –COCH3), 3.94 (2H, t, J = 6.5 Hz, –CH2(CH2)6CH3), 6.43
(1H, d, J = 15.5 Hz, H-8), 6.87 (2H, d, J = 8.5 Hz, H-30,50), 7.20 (1H,
d, J = 8.0 Hz, H-5), 7.38 (2H, m, H-2,6), 7.50 (2H, d, J = 8.5 Hz,
H-20,60), 7.66 (1H, d, J = 15.5 Hz, H-7). 13C NMR (CDCl3, 125 MHz)
d 14.1 (–(CH2)7CH3), 20.6 and 22.6 and 26.0 and 29.2 and 29.3
and 31.8 (–CH2(CH2)6CH3 and 2 ꢀ –COCH3), 68.3 (–OCH2(CH2)6-
CH3), 114.8 (C-30,50), 121.6 (C-20,60), 122.3 (C-8), 122.4 (C-1),
123.8 (C-2), 126.3 (C-5), 130.9 (C-10), 122.8 (C-6), 139.7 (C-7),
142.3 (C-4), 142.9 (C-3), 156.1 (C-40), 163.3 (C-9), 168.2
(2 ꢀ –COCH3).
8), 7.72 (2H, br s, H-5,6), 6.77 (2H, d, J = 8.5 Hz, H-30,50), 6.93 (1H,
br s, H-2), 7.38 (1H, d, J = 15.5 Hz, H-7), 7.39 (2H, d, J = 8.5 Hz,,
H-20,60), and NH signals. 13C NMR (CDCl3, 125 MHz) d 14.2
(–(CH2)7CH3), 22.7 and 26.1 and 26.6 and 28.4 and 29.3 and 29.5
and 29.8 and 31.9 (–CH2(CH2)6CH3 and Boc-CH3 and C-b and
C-c), 39.5 (C-d), 53.5 (C-a), 68.2 (–OCH2(CH2)6CH3), 79.6 (Boc-C),
114.3 (C-30,50), 115.5 (C-2, 8), 116.7 (C-6), 122.3 (C-20,60,5), 126.9
(C-1), 130.1 (C-10), 142.2 (C-7), 144.1 (C-4), 146.6 (C-3), 156.0
(C-40), 156.8 (Boc-CO), 167.6 (C-9), 171.1 (Orn-CO). ESI-MS (Posi-
tive): 598.2 ([M+H]ꢁ, 100%); ESI-MS (Negative): 632.6 ([M+Cl]ꢁ,
100%). HR-FAB-MS [M+H]+ m/z 598.35019 (calcd for C33H48O7N3,
requires 598.34922).
4.4.2. Compound 2c
Yield: 97%, 1H NMR (CDCl3 + CD3OD) d 0.89 (3H, t, J = 6.5 Hz,
–(CH2)7CH3), 1.30 (8H, m) and 1.44 (2H, m) and 1.77 (2H, m)
(–CH2(CH2)6CH3), 3.93 (2H, t, J = 6.5 Hz, –CH2(CH2)6CH3), 6.43
(1H, d, J = 15.5 Hz, H-8), 6.80 (1H, d, J = 7.5 Hz, H-6), 6.85 (2H, d,
J = 8.5 Hz, H-30,50), 6.92 (1H, d, J = 7.5 Hz, H-5), 7.05 (1H, br s,
H-2), 7.51 (3H, m, H-20,60,7). 13C NMR (CDCl3 + CD3OD, 125 MHz)
d 13.7 (–(CH2)7CH3), 22.4 and 25.8 and 29.0 and 29.1 and 31.6
(–CH2(CH2)6CH3), 68.1 (–OCH2(CH2)6CH3), 113.6 (C-2), 114.3
(C-30,50), 114.9 (C-6), 117.5 (C-8), 121.1 (C-5), 121.3 (C-20,60),
126.9 (C-1), 131.2 (C-10), 141.3 (C-7), 144.4 (C-4), 146.6 (C-3),
155.3 (C-40), 165.2 (C-9). ESI-MS (Positive): 384.2 ([M+H]ꢁ,
100%); ESI-MS (Negative): 382.3 ([MꢁH]ꢁ, 100%). HR-FAB-MS
[M+H]+ m/z 384.21764 (calcd for C23H30O4N, requires 384.21748).
4.3.3. Compound 3dp
After synthesis from 3a and purification on a SiO2 column, 3dp
was eluted out with hexane–EtOAc (1:1) (yield: 89%). 1H NMR
(CDCl3)
J = 6.5 Hz, H-
d
0.89 (3H, t, J = 6.5 Hz, –(CH2)7CH3), 1.08 (3H, d,
), 1.31 (8H, m) and 1.43 (2H, m) and 1.76 (2H, m)
c
(–CH2(CH2)6CH3), 1.38 (9H, s, tBu-CH3), 2.28 (3H, s) and 2.29 (3H,
s) (2 ꢀ –COCH3), 3.90 (2H, t, J = 6.5 Hz, –CH2(CH2)6CH3), 4.35 (1H,
m, H-b), 4.63 (1H, br s, H-a), 6.54 (1H, d, J = 15.5 Hz, H-8), 6.85
(1H, d, J = 8.5 Hz, H-30,50), 7.19 (2H, m, H-2,5), 7.41 (1H, dd,
J = 2.0, 8.5 Hz, H-6), 7.39 (2H, d, J = 8.5 Hz, H-20,60), 7.57 (1H, d,
J = 15.5 Hz, H-7). 13C NMR (CDCl3, 125 MHz) d 14.0 (–(CH2)7CH3),
16.8 and 20.4 and 20.5 and 22.5 and 25.9 and 28.0 and 29.1 and
29.2 and 31.7 (–CH2(CH2)6CH3, tBu-CH3 and 2 ꢀ –COCH3), 57.8
(C-
a
), 66.4 (C-b), 68.1 (–OCH2(CH2)6CH3), 114.7 (C-30,50), 121.1
4.4.3. Compound 3d
(C-20,60), 121.4 (C-6), 122.3 (C-8), 123.7 (C-2), 126.1 (C-5), 130.4
(C-1), 133.5 (C-10), 139.3 (C-7), 142.2 (C-4), 142.9 (C-3), 155.8
(C-40), 165.2 (C-9), 167.2 (Thr-CO), 167.9 (2 ꢀ –COCH3).
Synthesis from 3dp (yield: 89%), 1H NMR (CDCl3) d 0.88 (t,
J = 6.5 Hz, 3H, –(CH2)7CH3), 1.07 (3H, d, J = 6.0 Hz, H-Thr-CH3),
1.34 (m, 17H) and 1.61 (m, 2H) and 1.77 (m, 2H) (–CH2(CH2)6CH3
and H-t-Bu), 3.78 (2H, br s, –CH2(CH2)6CH3), 4.35 (1H, m, H-b),
4.3.4. Compound 4dp
4.62 (1H, t, J = 5.0 Hz, H-a), 6.31 (1H, d, J = 15.5 Hz, H-8), 6.88
After synthesis from 4a and purification on a SiO2 column, 4dp
(3H, m, H-6, 30,50), 6.94 (1H, d, J = 8.0 Hz, H-5), 7.02 (1H, d,
J = 3.0 Hz, H-2), 7.38 (2H, d, J = 8.5 Hz, H-20,60), 7.54 (1H, d,
J = 15.5 Hz, H-7) and some NH signals. 13C NMR (CDCl3, 125 MHz)
d 14.2 (–(CH2)7CH3), 17.7 and 18.2 and 22.9 and 26.3 and 28.4
and 29.5 and 29.6 and 32.7 (–CH2(CH2)6CH3 and tBu-CH3 and
was eluted out with hexane–ethyl acetate (1:1) (yield: 90%). [a]
D
+20.9 (c 0.2, MeOH); 1H NMR (CDCl3) d 0.88 (3H, t, J = 6.5 Hz,
–(CH2)7CH3), 1.29 (8H, m) and 1.45 (11H, m) and 1.76 (2H, m)
(–CH2(CH2)6CH3 and t-Bu), 2.30 (6H, s) (2 ꢀ –COCH3), 2.69 (1H, dd,
J = 7.0, 16.5 Hz) and 2.97 (1H, dd, J = 4.5, 16.5 Hz) (H-b), 3.90 (2H, t,
Thr-CH3), 58.4 (C-a), 67.2 (C-b), 68.6 (–OCH2(CH2)6CH3), 76.4
J = 6.5 Hz, –CH2(CH2)6CH3), 5.00 (1H, m, H-
a
), 6.40 (1H, d,
(C-t-Bu), 114.5 (C-30,50), 115.1 (C-2), 115.7 (C-8), 117.0 (C-6),
122.0 (C-20,60,5), 127.3 (C-1), 130.4 (C-10), 142.8 (C-7), 144.6
(C-4), 147.2 (C-3), 156.5 (C-40), 167.6 (C-9), 168.3 (Thr-CO). ESI-
MS (Positive): 541.2 ([M+H]ꢁ, 100%); ESI-MS (Negative): 575.5
([M+Cl]ꢁ, 100%). HR-FAB-MS [M+H]+ m/z 541.29310 (calcd for
J = 15.5 Hz, H-8), 6.83 (2H, d, J = 8.5 Hz, H-30,50), 7.20 (1H, d,
J = 8.0 Hz, H-5), 7.26 (1H, d, J = 2.0 Hz, H-2), 7.33 (1H, dd, J = 2.0,
8.0 Hz, H-6), 7.39 (2H, d, J = 8.5 Hz, H-20,60), 7.59 (1H, d, J = 15.5 Hz,
H-7). 13C NMR (CDCl3, 125 MHz) d 14.1 (–(CH2)7CH3), 20.6 and
22.6 and 26.0 and 28.0 and 29.2 and 29.3 and 31.8 (–CH2(CH2)6-
C31H45O6N2, requires 541.32775).
CH3, t-Bu-CH3 and 2 ꢀ –COCH3), 36.5 (C-b), 50.2 (C-
a), 68.1 (–OCH2-
(CH2)6CH3), 82.1 (tBu-C), 114.7 (C-30,50), 120.9 (C-6), 121.6 (C-20,60),
122.5 (C-8), 123.9 (C-2), 126.3 (C-5), 130.5 (C-1), 133.4 (C-10), 140.3
(C-7), 142.4 (C-4), 143.2 (C-3), 156.0 (C-40), 165.7 (C-9), 168.1 (Asp-
CONH, 2 ꢀ –COCH3), 171.8 (Asp-COOtBu).
4.4.4. Compound 4d
Synthesis from 4dp (yield: 95%). 1H NMR (CDCl3) d 0.87 (t,
J = 6.5 Hz, 3H, –(CH2)7CH3), 1.27 (m, 8H) and 1.32 (m, 11H) and
1.67 (m, 2H) (–CH2(CH2)6CH3 and t-Bu), 2.87 (2H, m, H-b), 3.81
(2H, t, J = 6.5 Hz, –CH2(CH2)6CH3), 5.11(1H, m, H-a), 6.16 (1H, d,
4.4. General method for the synthesis of 1d, 2c, 3d and 4d
J = 15.5 Hz, H-8), 6.68 (4H, m, H-5, 6, 30,50), 6.88 (1H, br s, H-2),
7.29 (2H, d, J = 8.5 Hz, H-20,60), 7.34 (1H, d, J = 15.5 Hz, H-7), and
some NH signals. 13C NMR (CDCl3, 125 MHz) d 14.1 (–(CH2)7CH3),
22.6 and 26.0 and 28.0 and 29.2 and 29.4 and 31.8 (–CH2(CH2)6CH3
Compound 1dp, 2p, 3dp or 4dp (0.5 mmol) was dissolved in
THF (1.5 ml) and MeOH 1.5 ml. To the mixture was added 0.2 M
KOH (H2O–MeOH 9:1 solution, 0.8 ml). The mixture was stirred
at rt for 1 h followed by the addition of 1 M HCl (0.5 ml). The mix-
and tBu-CH3), 37.4 (C-b), 50.5 (C-
a), 68.2 (–OCH2(CH2)6CH3), 82.3
(tBu-C), 114.0 (C-2), 114.6 (C-30,50), 115.4 (C-8), 116.4 (C-6),

7014
C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 7009–7014
122.4 (C-20,60,5), 126.9 (C-1), 129.9 (C-10), 142.7 (C-7), 144.1 (C-4),
146.8 (C-3), 156.4 (C-40), 167.5 (C-9), 169.5 (ASP-COOtBu), 171.1
(Asp-CONH). ESI-MS (Positive): 555.2 ([M+H]ꢁ, 100%). HR-FAB-
MS [M+H]+ m/z 555.30644 (calcd for C31H43O7N2, requires
555.30703).
147.0 (C-3), 156.1 (C-40), 167.3 (C-9), 168.8 (ASP-CONH), 173.6
(ASP-COOH). ESI-MS (Positive): 499.1 ([M+H]ꢁ, 100%); ESI-MS
(Negative): 497.1 ([MꢁH]ꢁ, 100%). HR-FAB-MS [M+H]+ m/z
499.24404 (calcd for C27H35O7N2, requires 499.24443).
4.6. 1,3-b-D-Glucan synthase assay
4.5. General method for the synthesis of 1c, 3c and 4c
The fungal membrane enzyme was prepared from Candida albi-
cans using a previously reported procedure.6,7 The 1,3-b-
-glucan
synthase reaction mixture contained 50 mM Tris–HCl (pH 7.5),
0.5 mM EDTA, 25 mM KF, 20 M GTP -S, 0.75% BSA, 5.0 mM
UDP-Glc and the membrane-bound 1, 3-b- -glucan synthase. The
Compound 1d, 3d or 4d (0.5 mmol) was treated with 90% TFA at
rt for 30 min. The mixture was concentrated to dryness to obtain
1c, 3c or 4c.
D
l
c
D
4.5.1. Compound 1c
reaction was initiated by adding of the enzyme, reacted for
20 min by incubation at 30 °C and was terminated by heating at
96 °C for 10 min. The 1,3-b-D-glucan-dependent enzyme cascade
Yield: 92%, 1H NMR (CDCl3 + CD3OD) d 0.87 (3H, t, J = 6.5 Hz,
–(CH2)7CH3), 1.26 (8H, m) and 1.38 (2H, m) and 1.69 (5H, m) and
1.89 (1H, m) (–CH2(CH2)6CH3 and H-b and H-
c), 2.90 (2H, br s,
reaction was carried out using a b-glucan determination kit
H-d), 3.84 (2H,t, J = 6.5 Hz, –CH2(CH2)6CH3), 4.59 (1H, br s, H-
a
),
(BGSTAR) following the procedure attached to the kit.
6.28 (1H, d, J = 15.5 Hz, H-8), 6.69 (2H, br s, H-5,6), 6.75 (2H, d,
J = 8.5 Hz, H-30,50), 6.97 (1H, br s, H-2), 7.28 (1H, d, J = 15.5 Hz,
H-7), 7.35 (2H, d, J = 8.5 Hz, H-20,60), and some OH and NH signals.
13C NMR (CDCl3 + CD3OD, 75 MHz) d 14.0 (–(CH2)7CH3), 22.6 and
23.6 and 26.0 and 29.2 and 29.3 and 31.8 (–CH2(CH2)6CH3 and
4.7. Anti-fungal assay
The anti-fungal activities were determined by broth dilution
method according to NCCLS using Candida albicans NBRC 1594 (from
NITE, Tokyo, Japan) as the organism.5 The concentration of the
C-b and C-c), 39.1 (C-d), 53.4 (C-a), 68.2 (–OCH2(CH2)6CH3),
113.8 (C-2), 114.4 (C-30,50), 115.3 (C-6), 116.2 (C-8), 122.0
(C-20,60), 122.1 (C-5), 126.7 (C-1), 129.9 (C-10), 142.1 (C-7), 144.3
(C-4), 146.8 (C-3), 156.0 (C-40), 167.8 (C-9), 170.2 (Orn-CO). ESI-
MS (Positive): 498.3 ([M+H]ꢁ, 100%); ESI-MS (Negative): 532.6
([M+Cl]ꢁ, 100%). HR-FAB-MS [M+H]+ m/z 498.29615 (calcd for
anti-fungal agents was in the range between 0.12 and 64 lg/ml.
Acknowledgments
We are grateful to Dr. Yue-Zhong Shu of Bristol-Myers Squibb
Research and Development, Wallingford, CT, USA, for his important
suggestions to carry out this research. We would also like to thank
Dr. Masahiko Miyamoto of the Faculty of Pharmaceutical Sciences,
Niigata University of Pharmacy and Applied Life Sciences for his
valuable advices during the preparation of the fungal membrane
enzyme.
C28H40O5N3, requires 498.29679).
4.5.2. Compound 3c
Yield: 98%, 1H NMR (CDCl3 + CD3OD) d 0.88 (3H, t, J = 6.5 Hz,
–(CH2)7CH3), 1.23 (3H, d, J = 6.5 Hz, H-
(2H, m) and 1.71 (2H, m) (–CH2(CH2)6CH3), 3.86 (2H, t, J = 6.5 Hz,
–CH2(CH2)6CH3), 4.36 (1H, m, H-b), 4.65 (1H, br s, H- ), 6.32 (1H,
c), 1.28 (8H, m) and 1.40
a
d, J = 15.5 Hz, H-8), 6.74 (1H, d, J = 8.5 Hz, H-5), 6.78 (3H, m,
H-30,50,6), 6.99 (1H, br s, H-2), 7.38 (2H, d, J = 8.5 Hz, H-20,60),
7.42 (1H, d, J = 15.5 Hz, H-7). 13C NMR (CDCl3 + CD3OD, 125 MHz)
d 13.9 (–(CH2)7CH3), 18.5 and 22.5 and 25.9 and 29.1 and 29.2
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.08.022.
and 31.7 (–CH2(CH2)6CH3 and C-c), 58.3 (C-a), 67.3 (C-b), 68.2
(–OCH2(CH2)6CH3), 113.8 (C-2), 114.6 (C-30,50), 115.2 (C-6), 116.4
(C-8), 122.0 (C-20,60), 122.2 (C-5), 126.8 (C-1), 129.9 (C-10), 142.5
(C-7), 144.5 (C-4), 146.9 (C-3), 156.2 (C-40), 168.0 (C-9), 169.2
(Thr-CO). ESI-MS (Positive): 485.1 ([M+H]ꢁ, 100%). HR-FAB-MS
[M+H]+ m/z 485.26525 (calcd for C27H37O6N2, requires 485.26517).
References and notes
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4. Lafay, S.; Gil-Izquierdo, A.; Manach, C.; Morand, C.; Besson, C.; Scalbert, A. J.
Nutr. 2006, 136, 1192.
5. NCCLS, reference method for broth dilution antifungal susceptibility testing of
yeasts; approved standard- second edition, M27-A2, National Committee for
Clinical Laboratory Standards, Villanova, PA 2003.
6. Cabib, E.; Kang, M. S. Methods Enzymol. 1987, 138, 637.
7. Selvakumar, D.; Miyamoto, M.; Furuichi, Y.; Komiyama, T. Antimicrob. Agents
Chemother. 2006, 50, 3090.
8. Zeng, K.; Thompson, K. E.; Yates, C. R.; Miller, D. D. Bioorg. Med. Chem. Lett. 2009,
195, 458.
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Herscovici, J.; Scherman, D. Bioorg. Med. Chem. Lett. 2005, 15, 3224.
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Lemke, T. L., Williams, D. A., Roche, V. F., Zito, S. W., Eds., 6th ed.; Lippincott
Williams & Wilkins: Philadephia, 2008; pp 284–285.
4.5.3. Compound 4c
Yield: 98%, 1H NMR (CDCl3 + CD3OD) d 0.88 (3H, t, J = 6.5 Hz,
–(CH2)7CH3), 1.28 (8H, m) and 1.41 (2H, m) and 1.75 (2H, m)
(–CH2(CH2)6CH3), 2.89 (m, partially overlapped with H2O signal,
H-b), 3.91 (2H, t, J = 6.5 Hz, –CH2(CH2)6CH3), 5.03 (1H, t, d,
J = 6.5 Hz, H-a), 6.30 (1H, d, J = 15.5 Hz, H-8), 6.82 (3H, m,
H-30,50,5), 6.91 (1H, dd, J = 1.5, 8.0 Hz, H-6), 7.04 (1H, 1H, d,
J = 1.5 Hz, H-2), 7.40 (2H, d, J = 8.5 Hz, H-20,60), 7.49 (1H, d,
J = 15.5 Hz, H-7). 13C NMR (CDCl3 + CD3OD, 125 MHz)
d
14.0
(–(CH2)7CH3), 16.9 and 22.5 and 25.9 and 29.1 and 29.2 and 31.7
(–CH2(CH2)6CH3), 35.7 (C-b), 50.0 (C-a), 68.2 (–OCH2(CH2)6CH3),
113.5 (C-2), 114.6 (C-30,50), 115.1 (C-6), 116.4 (C-8), 121.8
(C-20,60,5), 126.8 (C-1), 130.3 (C-10), 142.6 (C-7), 144.6 (C-4),