Total synthesis and further scrutiny of the in vitro antifungal activity of 6-nonadecynoic acid

Article abstract of DOI:10.1002/ardp.200500102

The antifungal, naturally occurring acetylenic fatty acid 6-nonadecynoic acid was synthesized in three steps (18% overall yield), for the first time starting with commercially available 1-tetradecyne. The synthesis developed herein will facilitate the further study of the antifungal properties of this naturally occurring acetylenic fatty acid. The 6-nonadecynoic acid exhibited the best antifungal activity (< 4.3 μM) against Cryptococcus neoformans ATCC 66031 in Sabouraud Dextrose Broth (SDB) media. In our hands, it was not active against Candida albicans ATCC 14053 and Candida albicans ATCC 60193.

Full text of DOI:10.1002/ardp.200500102

Arch. Pharm. Chem. Life Sci. 2005, 338, 441−443
DOI 10.1002/ardp.200500102
441
Total Synthesis and Further Scrutiny of the in vitro
Antifungal Activity of 6-Nonadecynoic Acid
a
a
b
´
Nestor M. Carballeira , David Sanabria , Keykavous Parang
a Department of Chemistry, University of Puerto Rico, San Juan, Puerto Rico
b Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode
Island, Kingston, USA
The antifungal, naturally occurring acetylenic fatty acid 6-nonadecynoic acid was synthesized in three
steps (18% overall yield), for the first time starting with commercially available 1-tetradecyne. The
synthesis developed herein will facilitate the further study of the antifungal properties of this naturally
occurring acetylenic fatty acid. The 6-nonadecynoic acid exhibited the best antifungal activity
(< 4.3 µM) against Cryptococcus neoformans ATCC 66031 in Sabouraud Dextrose Broth (SDB) media.
In our hands, it was not active against Candida albicans ATCC 14053 and Candida albicans ATCC
60193.
Keywords: Synthesis; Fatty acids; Antifungal; Acetylenic acids; C. neoformans
Received: February 28, 2005; Accepted: April 1, 2005
Introduction
The antimicrobial and antifungal properties of acetylenic
fatty acids have been an active area of lipid research for
some time [1Ϫ3]. Just recently, a novel acetylenic fatty acid,
6-nonadecynoic acid 1, together with the known 6-octade-
cynoic acid, was isolated from an ethanol extract of the
Scheme 1. Synthesis route for 6-nonadecynoic acid.
roots of Pentagonia gigantifolia [4]. The acetylenic acid 1
was reported to be fungistatic and specific to fluconazole-
susceptible and resistant Candida albicans strains with MIC
values (0.52 µg/mL) comparable to those of amphotericin B
(0.52 µg/mL) and fluconazole (0.29 µg/mL) [4]. The anti-
fungal mechanism of action of 1 has been suggested to be
due to the interference of the compound with fungal sphin-
golipid biosynthesis [4]. Therefore, the nonadecynoic acid 1
is a potential antifungal lead substance for further studies.
The synthesis of large quantities of 1 is needed for these
studies. Herein, we report the first total synthesis of 1 and,
in addition, we report that 1 is also fungistatic to a strain
of Cryptococcus neoformans but not effective against other
strains of Candida albicans. This finding is in contrast to
what was previously reported for the 6-nonadecynoic acid
(1), which was found to be specific towards C. albicans but
not fungistatic towards C. neoformans [4].
Reagents and conditions: i Br-(CH2)5OTHP, n-BuLi, THF,
HMPA, Ϫ10°C; ii PTSA, MeOH, 45°C; iii PDC, DMF, rt.
cyne (2) which was successfully alkylated with 2-(5-bromo-
pentyloxy)tetrahydropyran in the presence of n-BuLi in
THF-HMPA at Ϫ10°C (better solubility at this tempera-
ture) which resulted in the tetrahydropyranyl protected al-
kyne 3 in 82% yield (Scheme 1). Deprotection of 3 was
achieved with p-toluenesulfonic acid (PTSA) in methanol at
45°C, which yielded the desired 6-nonadecyn-1-ol (4) in a
45% yield. Final oxidation of 4 with pyridinium dichromate
(PDC) in DMF afforded the 6-nonadecynoic acid (1) in
49% isolated yield. The overall yield for this three-step syn-
thesis was 18%. The synthetic acid 1 presented spectral data
identical with the natural acetylenic fatty acid 1 as pre-
viously reported [4].
Results and discussion
The antifungal activity of 1 against Cryptococcus neo-
formans ATCC 66031, Candida albicans ATCC 14053,
Candida albicans ATCC 60193, and Aspergillus niger ATCC
16404 in SDB media were determined using a modified
method of the National Committee for Clinical Laboratory
Standards (NCCLS) as described by Galgiani and the more
recent NCCLS M27-A microdilution methods as described
previously [5-8]. As shown in Table 1 amphotericin B and
Our approach towards the total synthesis of 6-nonadecy-
noic acid (1) started with commercially available 1-tetrade-
´
Correspondence: Nestor M. Carballeira, Department of Chemistry,
University of Puerto Rico, PO Box 23346, San Juan, Puerto Rico
00931-3346. Phone ϩ1809 787 764-0000, ext. 4791, Fax: ϩ1809 787
756-8242, e-mail: ncarball@upracd.upr.clu.edu
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

442
Carballeira et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 441−443
Table 1. Antifungal activity (MIC values, µM)† against Cryptococcus neoformans (SDB), Candida albicans (SDB), and
Aspergillus niger (SDB) at 35Ϫ37°C after 24Ϫ48 h.
Compound
C. neoformans
ATCC 66031
C. albicans
ATCC 14053
C. albicans
ATCC 60193
A. niger
ATCC 16404
1
< 4.3
< 1.9
< 0.2
6672
4003
< 0.2
> 5000
8896
> 4003
< 0.2
2224
1001
< 0.2
> 5000
Fluconazole
Amphotericin B
DMSO
> 5000
> 5000
†
The result is the average of three separate experiments.
million (ppm) relative to CDCl3 (77.0 ppm). Mass spectra data was
acquired using a GC-MS (Hewlett-Packard 5972A MS Chem-
Station; Hewlett-Packard, Palo Alto, CA, USA) at 70 eV equipped
with a 30 m ϫ 0.25 mm special performance capillary column (HP-
5MS) of polymethylsiloxane cross-linked with 5% phenyl methyl-
polysiloxane.
fluconazole were used as positive controls. Acid 1 displayed
antifungal activity against C. neoformans ATCC 66031 with
a MIC of less than 4.3 µM but did not exhibit any activity
against A. niger ATCC 16404 (MIC 2.224 µM) and, more
surprisingly, neither against Candida albicans ATCC 14053
(MIC 6.672 µM) nor Candida albicans ATCC 60193 (MIC
8.896 µM). The two latter C. albicans strains are fluconazole
resistant strains. It was previously reported that the natural
acetylenic fatty acid 1 is specific towards C. albicans ATCC
90028 but not active against C. neoformans. This difference
in fungitoxicity could be due to differences in the suscepti-
bility of C. neoformans and C. albicans strains. Nevertheless,
our results underline once again the importance of acid 1
as an antifungal compound.
1-[(Tetrahydropyran-2-yl)oxy]-6-nonadecyne (3)
To a stirred solution of 1-tetradecyne (0.82 g, 4.2 mmol) in dry THF
(5.0 mL), n-BuLi (2.5 M, 10.0 mmol) in dry hexane (4.0 mL) was
added dropwise at room temperature. After 80 min, HMPA (10.0
mL) and 2-(5-bromopentyloxy)tetrahydropyran (1.06 g, 4.2 mmol)
were added dropwise to the reaction mixture while maintaining the
temperature approximately at Ϫ 60°C. After 24 h, the reaction mix-
ture was poured into a large volume of water and extracted with
hexane (2 ϫ 20 mL). The organic layer was washed with brine
(1 ϫ 20 mL) before drying over MgSO4. Filtration, evaporation of
the solvent and fractional distillation furnished 1.25 g of 1-[(tetra-
hydropyran-2-yl)oxy]-6-nonadecyne (3) in 82% yield. 1H-NMR
(500 MHz, CDCl3) δ 4.57 (1H, t, J ϭ 2.3 Hz), 3.86Ϫ3.73 (2H,
m), 3.49-3.38 (2H, m), 2.12 (4H, m, H-5, H-8), 1.81Ϫ1.68 (6H, m),
1.61Ϫ1.41 (8H, m), 1.24 (18H, m, -CH2-), 0.87 (3H, t, J ϭ 6.9 Hz,
H-19); 13C-NMR (75 MHz, CDCl3) δ 98.8 (d), 80.4 (s), 79.9 (s),
67.4 (t, C-1), 62.3 (t), 31.9 (t, C-17), 30.7 (t, C-2), 29.6 (t), 29.5 (t),
29.32 (t), 29.27 (t), 29.1 (t), 29.0 (t), 28.9 (t), 28.5 (t), 25.5 (t), 22.7
(t, C-18), 19.6 (t, C-3), 18.7 (t), 18.4 (t), 14.1 (q, C-19); GC-MS m/z
(% rel. int.): [M]ϩ 364 (1), 291 (4), 279 (2), 209 (2), 195 (8), 181 (1),
167 (1), 165 (1), 151 (1), 148 (1), 135 (2), 125 (3), 123 (3), 111 (4),
109 (9), 101 (10), 97 (5), 95 (14), 85 (100), 81 (14), 79 (12), 67 (22),
57 (12), 55 (19).
In summary, we have accomplished the first total synthesis
of the 6-nonadecynoic acid 1, a novel potent antifungal ace-
tylenic fatty acid. Our synthetic approach should provide
access to considerable amounts of 1 for further biological
evaluation. In addition, we have shown that 1 is indeed anti-
fungal against C. neoformans at concentrations that are
comparable with those of fluconazole, but in contrast to
what was previously reported, it is active only against some
strains of C. albicans.
Acknowledgments
This work was supported by a grant from the SCORE pro-
gram of the National Institutes of Health (Grant No.
S06GM08102). D. Sanabria thanks the NIH-RISE program
for a graduate fellowship. This research was also supported
in part by NIH Grant Numbers P20RR16457 and
P20RR16470 from the BRIN/INBRE program of the
National Center for Research Resources.
6-Nonadecyn-1-ol (4)
1-[(Tetrahydropyran-2-yl)oxy]-6-nonadecyne (1.4 g, 3.9 mmol) in
methanol (10.0 mL), and catalytic amounts of p-toluene sulfonic
acid (PTSA) were stirred at 45°C for 3 h. The solvent was evapo-
rated, hexane (5 mL) and diethyl ether (5 mL) were added to crys-
tallize excess PTSA. Afterwards it was filtered and evaporated under
high vacuum affording 0.05 g (45%) of 6-nonadecyn-1-ol (4). This
product was used in the next step without further purification. IR
(neat) νmax 3314 (br), 2925, 2854, 2116, 1463, 1234, 1186, 1136, 1051
cmϪ1 1H-NMR (300 MHz, CDCl3) δ 3.64 (2H, t, J ϭ 6.5 Hz, H-
;
Experimental
1), 2.31 (1H, br, -OH), 2.14 (4H, m, H-5, H-8), 1.55 (2H, quintet,
J ϭ 6.7 Hz, H-2), 1.45 (4H, quintet, J ϭ 6.9 Hz, H-4, H-9), 1.25
(20 H, m, -CH2-), 0.87 (3H, t, J ϭ 6.8 Hz, H-19); 13C-NMR (75
MHz, CDCl3) δ 80.5 (s), 79.8 (s), 62.9 (t, C-1), 32.2 (t), 31.9 (t),
29.6 (t), 29.5 (t), 29.3 (t), 29.1 (t), 28.9 (t), 24.9 (t, C-3), 22.7 (t, C-
18), 18.72 (t), 18.70 (t), 14.1 (q, C-19); GC-MS m/z (% rel. int.):
[Mϩ1]ϩ 281 (1), 180 (2), 166 (2), 149 (3), 135 (8), 121 (10), 108 (34),
93 (66), 82 (100), 67 (83), 55 (76).
IR spectra were recorded on a Nicolet 600 FT-IR spectrophoto-
meter (Thermo-Nicolet, Madison, WI, USA). 1H- and 13C-NMR
spectra were recorded on a General Electric QE-300, Bruker DPX-
300 or on a Bruker DRX-500 spectrometer (General Electric Mag-
netic Resonance, Fremont, CA, USA or Bruker-Biospin, Billerica,
MA, USA). 1H-NMR chemical shifts are reported with respect to
internal Me4Si and 13C chemical shifts are reported in parts per
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 441−443
6-Nonadecynoic acid (1)
Synthesis and Fungitoxicity of 6-Nonadecynoic Acid
443
of DMSO in the assays was 5% (v/v). DMSO was not inhibitory to
the organisms in the concentrations tested.
To a stirred solution of 6-nonadecyn-1-ol (0.056 g, 0.20 mmol) in
3.0 mL DMF pyridinium dichromate (0.3 g, 0.80 mmol) was slowly
added at room temperature. After 24 h, the reaction mixture was
poured in 10 mL of water and extracted with hexane (3 ϫ 10 mL).
Once the solvent was evaporated, the mixture was dried under vac-
uum and impurities were vacuum distilled (Kugelrohr short-path
distillation). Thereby, 0.029 g of 9-nonadecynoic acid (1) was ob-
tained, resulting in a 49% yield. Acid 1 presented identical spectral
data with the natural acetylenic acid 1 previously reported, and the
purity of the compound was estimated to be >97% by 13C-NMR
[4].
Susceptibility testing
Microdilutions for control experiments with C. albicans, A. niger,
and C. neoformans were performed according to the modified
method of National Committee for Clinical Laboratory Standards
(NCCLS) method as described by Galgani [5] and according to the
more recent NCCLS M27-A microdilution methods as described
previously [7Ϫ8]. Dilutions were prepared in 0.1 mL of SDB; the
inocula were either 104 C. albicans or C. neoformans cells or A. niger
spores. The tubes were incubated for 24-48 h at 36
1°C, and
turbidity was read visually. MICs were calculated in comparison to
growth control as the lowest concentration that shows inhibition for
AMB, FLC, and the test compounds.
Microorganisms
A. niger ATCC 16404, C. neoformans ATCC 66031, C. albicans
ATCC 14053, and C. albicans ATCC 60193 were obtained from
American Type Culture Collection, Manassas, VA, USA. Stock cul-
tures were kept on Sabouraud Dextrose Agar (SDA; Becton-Dickin-
son and Co., Sparks, MD, USA). Subcultures were prepared on
SDA at 35-37°C. Suspension cultures were prepared by inoculation
of single colonies in 7 ml of normal saline solution. Prior to prep-
aration of susceptibility assays, yeast cells were resuspended in nor-
mal saline to yield a transmittance of 73-75% at 530 nm that equals
a concentration of 106 cells/mL and spores of A. niger in saline
medium produce a similar transmittance at 530 nm compared to
the control tube. The media was Sabourad Dextrose Broth (SDB).
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 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim