Antifungal activity of bicyclic monoterpenoids and terpenesulfides

Article abstract of DOI:10.1007/s10600-010-9517-5

A total of 33 compounds including bicyclic monoterpenes, oxides, allyl alcohols, and S-containing pinane and carane derivatives were screened for antifungal activity against mycelial and yeast-like fungi. The structure-activity relationship was studied. The most active antimycotics were found.

Full text of DOI:10.1007/s10600-010-9517-5

Chemistry of Natural Compounds, Vol. 46, No. 1, 2010
ANTIFUNGALACTIVITY OF BICYCLIC MONOTERPENOIDS
AND TERPENESULFIDES
1*
1
1
L. E. Nikitina, V. A. Startseva, L. Yu. Dorofeeva,
UDC 547.598.3+546.271
1
1
N. P. Artemova, I. V. Kuznetsov,
2
2
S. A. Lisovskaya, and N. P. Glushko
A total of 33 compounds including bicyclic monoterpenes, oxides, allyl alcohols, and S-containing pinane
and carane derivatives were screened for antifungal activity against mycelial and yeast-like fungi. The
structure–activity relationship was studied. The most active antimycotics were found.
Keywords: carane and pinane monoterpenoids, antifungal activity.
We performed for the first time a systematic study of the antifungal activity of series of pinane and carane
monoterpenoids with a broad set of S-containing functional groups and studied the structure–activity relationship to find the
most active antimycotics.
At the start of our work, the literature contained data on the antifungal activity of ꢀ- and ꢁ-pinenes in addition to the
antifungal effect of essential oils containing (+)-3-carene (1), verbenols, and myrtenol [1–5]. However, the antifungal activity
of S-containing terpenoids was not previously investigated.
Carane thioterpenoids 4–15 were synthesized by reacting stereoisomeric 3-carene oxides (2 and 3) with thiols (4, 5,
10, 11, 15), isothiuronium salts (6–9, 13, 14), and thiourea (16) in the presence of sodium ethoxide [6–9]. Sulfoxide 12 was
obtained by oxidation of 6 with H O in glacial acetic acid. The products were isolated by column chromatography. The
2
2
structures were established using spectral data.
O
OH
O
R
RS
H
S
OH
OH
O
H
SR
H
1
2
3
4, 6, 8
5, 7, 9 - 11
12
R = CH (4, 5), CH CH=CH (6, 7, 12), CH Ph (8, 9), iso-Bu (10), n-Bu (11)
2
2
3
2
S
S
S
OH
HO
O
H
O
H
H
CH
H
2
H
OH
H
S
OH
OH
S
13
14
15
16
1) Kazan State Medical University, 420012, Republic of Tatarstan, Kazan, e-mail: nikit@mi.ru; 2) Kazan Institute of
Epidemiology and Microbiology, 420015, Republic of Tatarstan, Kazan, e-mail: farrus@mail.ru. Translated from Khimiya
Prirodnykh Soedinenii, No. 1, pp. 27–30, January–February, 2010. Original article submitted January 19, 2009.
©
28
0009-3130/10/4601-0028 2010 Springer Science+Business Media, Inc.

TABLE 1. Antifungal Activity of Carane Compounds
Compound
1 nonpath.
1 path.
2
3
4
5
6
7
8
9
1
2
3
4
5
6
3+
–
–
2+
+/–
–
2+
–
–
2+
+/–
–
2+
–
2+
+/–
+/–
–
2+
–
+
+/–
+/–
–
2+
–
–
2+
2+
–
2+
–
–
+
+/–
–
2+
+/–
2+
2+
+
2+
–
+
3+
+
–
+
–
+/–
+
+
–
2+
+/–
–
2+
2+
–
–
+
–
+/–
–
–
+/–
–
–
–
–
–
–
+/–
–
+/–
–
–
n.d.
–
+/–
–
+
+/–
+/–
+/–
+/–
n.d.
+/–
–
–
–
+
+/–
–
–
–
–
–
+
+/–
–
2+
–
–
+/–
2+
–
–
–
+/–
+/–
–
+
+
n.d.
–
–
–
–
+/–
+/–
+/–
–
+
–
+/–
–
–
–
–
–
–
–
–
–
–
+/–
–
–
–
n.d.
–
n.d.
–
–
–
+
–
7
8
9
10
11
12
13
14
15
16
–
n.d.
2+
n.d.
–
–
–
–
–
–
–
+/–
______
1 – Candida albicans, 2 – Candida parapsilosis, 3 – Rhodotorula rubra, 4 – Aspergillus niger, 5 – Penicillium tardum,
6 – Candida kruzei, 7 – Epidermophyton floccosum, 8 – Aspergillus fumigatus, 9 – Penicillium chrysogenum.
n.d. – no data.
(+)-3-Carene (1), 3-carene ꢀ-oxide (2), 3-carene ꢁ-oxide (3), and S-containing carane derivaties 4–16 were tested for
antifungal activity against mycelial and yeast-like fungi. Table 1 presents the test results. The “+” sign denotes a growth
inhibition zone of 1–3 mm (weak activity); “2+”, of 3–5 mm (moderate activity); “3+”, ꢂ5 mm (high activity). A lack of
activity is denoted by a “–” sign. The “+/–” sign denotes a growth inhibition zone of up to 1 mm (very weak activity). The
negative controls were plates without compounds that were treated in the same manner with solvent. The positive controls
were disks with Polisept “4+” fungicide.
Screening for antifungal activity of 16 carane compounds showed that (+)-3-carene (1) and 4ꢁ-methylthiocaran-3ꢀ-
ol (4) had the highest activity (Table 1). A comparison of the antifungal activity of 6, 7–9, 12–16 with that of starting oxides
2 and 3 showed that the antifungal effect of the terpenesulfides was lower whereas adding an S-CH group to 3-carene oxide
3
increased the antifungal activity of the thioterpenoids.
Thus, 4, which was synthesized from 3-carene ꢀ-oxide, exhibited moderate activity (2+) against Candida albicans
(nonpath.), C. albicans (path.), Aspergillus niger, C. krusei, and Penicillium chrysogenum and high activity (3+) against
Epidermophyton flocossum. Also, stereoisomer 5, which was prepared from 3-carene ꢁ-oxide, exhibited moderate activity
(2+) against only A. niger and P. chrysogenum. Thus, the steric structure of the molecule and the structure of the added group
affected the manifestation of antifungal activity by the compounds.
OH
HO
HO
OH
17
18
19
20
21
23
22
CH SR
2
CH SR
CH OH
2
2
H
RS
SR
24
25 - 28
29, 30
31, 32
33
R = i-Pr (25), CH CH SCH CH SH (26), CH COOH (27, 29), CH COOCH (28, 30, 32, 33), CH CH SH (31)
2
2
2
2
2
2
3
2
2
29

TABLE 2. Antifungal Activity of Pinane Compounds
Compound
1 nonpath.
1 path.
2
3
4
5
6
7
8
9
+/–
+
–
+/–
+/–
–
+
2+
+
–
2+
–
–
–
2+
+
–
–
–
–
+
2+
2+
–
+/–
–
–
–
–
+
2+
–
–
+
+/–
–
2+
3+
+/–
2+
n.d.
–
–
+/–
+/–
+
n.d.
2+
+
+
–
–
–
2+
+
2+
n.d.
+
–
2+
–
3+
n.d.
+
+
–
–
n.d.
–
3+
+/–
+/–
n.d.
+/–
–
+/–
+/–
–
–
n.d.
+
–
+/–
–
3+
2+
+/–
–
+/–
–
–
+/–
–
+/–
2+
n.d.
+/–
+/–
+/–
2+
2+
+
n.d.
2+
+
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
+
+
+
n.d.
3+
+/–
+
+/–
2+
–
+
–
+/–
–
2+
+/–
+/–
+/–
+/–
–
+/–
3+
+/–
+/–
–
–
+/–
+
–
+
+/–
+/–
+
–
n.d.
+/–
n.d.
n.d.
2+
–
2+
–
+/–
n.d.
+
–
+/–
n.d.
n.d.
3+
+
2+
n.d.
3+
+
n.d.
n.d.
+
+/–
+/–
–
+/–
+/–
–
+
______
1 – Candida albicans, 2 – Candida parapsilosis, 3 – Rhodotorula rubra, 4 – Aspergillus niger, 5 – Penicillium tardum,
6 – Candida kruzei, 7 – Epidermophyton floccosum, 8 – Aspergillus fumigatus, 9 – Penicillium chrysogenum.
n.d. – no data.
A comparison of the antifungal activity of structural isomers, 10 and 11, which contain butyl and iso-butyl groups,
found that 10 was better than 11 and had moderate activity against A. niger whereas 11 had weak antifungal activity. Thus, the
degree of branching in the hydrocarbon chain of the substituent affects the antifungal activity.
Pinane terpenesulfides 25–33 were synthesized by methods developed by us [10–12]. Allyl alcohols 20–22 and 24
were reacted with mono- and bifunctional thiols in the presence of ZnCl with replacement of the OH group by sulfide. This
2
produced 25–30 and 33. The main product in every instance from reactions of (–)-ꢁ-pinene (17) with thiols was the pinane
cis-isomer (31 and 32).
We also screened 17 pinane compounds for antifungal activity. These were bicyclic monoterpenes (–)-ꢁ-pinene (17),
(+)- and (–)-ꢀ-pinenes (18 and 19), allyl alcohols (+)- and (–)-cis-verbenols (20 and 21), (+)- and (–)-trans-verbenols (22 and
23), and (+)-myrtenol (24) and S-containing derivatives 25–33 prepared from them. They were tested against mycelial and
yeast-like fungi. Table 2 presents the test results.
If pinane compounds with a hydroxyl group such as cis- and trans-verbenols 20–23 are compared with ꢀ- and
ꢁ-pinenes 17–19, then an increase of antifungal activity is noted for the pinane alcohols. However, introducing a hydroxyl in
and of itself is not the only factor that helps to increase the antifungal activity of the molecules. The most active of all starting
monoterpenoids was (+)-trans-verbenol (22).
Introducing sulfide groups such as S–iPr, –S–(CH ) SH, S–(CH ) S(CH ) SH, and –S–CH –COOH into the pinane
2 2
2 2
2 2
2
structure produces compounds 25–27 and 29 with practically no antifungal activity.
A pinene terpenesulfide that had a methyl ester of mercaptoacetic acid (28) exhibited high antifungal activity (3+)
against A. niger, C. krusei, and P. chrysogenum.
Furthermore, the most active compounds 1, 4, 22, and 28 exhibited moderate activity against the especially dangerous
pathogenic fungus C. albicans, which causes serious systemic infections in man [13].
Carane and pinane monoterpenoids that were tested for antifungal activity were slightly toxic compounds
(LD = 950–5000 mg/kg) (mouse, i.p.). Studies of monoterpenoids for mutagenicity and genotoxicity indicated that they
50
were not direct mutagens. Therefore, there is little probability that they will exhibit carcinogenic properties.
30

EXPERIMENTAL
NMR spectra were measured in CDCl on a Varian Unity (300 and 75.43 MHz) spectrometer with TMS internal
3
standard; GC–MS, in a Turbo Mass Gold mass spectrometer (Perkin–Elmer) with a capillary column (30 m length, 320 ꢃm
diameter, ꢄ = 1.2 mL/min). Reaction products were isolated and purified using adsorption chromatography on silica gel
He
“L” (100/160 ꢃm). We used S-containing reagents from Aldrich. Terpene alcohols 20–24 were prepared at the Institute of
Chemistry, Komi Scientific Center, Ural Branch, RAS (Syktyvkar) [14]. Synthetic methods and spectral data of 4–15 [6–9],
of 25–28 [10, 11], and of 31 and 32 [12] have been published. Solvents were purified and dried according to the usual methods
[15].
Oxidation of Sulfide 6 to Sulfoxide 12. General Method. Sulfide 6 (0.07 g, 0.031 mol) and glacial acetic acid
(0.02 g, 0.031 mol) were stirred, cooled on ice, treated with H O (27.5%, 0.035 g, 0.031 mol), and left overnight at room
2
2
temperature. The absence of sulfide in the mixure signaled completion of the reaction. The mixture was diluted with H O and
2
extracted with CHCl (3 ꢅ 10 mL). The extract was washed with H O, aqueous K CO solution, and water again and dried
3
2
2
3
over MgSO . The product was purified by column chromatography (hexane:Et O, 5:1). Yield of 12, 75%.
4
2
3ꢀ-Hydroxy-4ꢁ-allylsulfinylcarane (12). PMR spectrum (ꢆ, ppm, J/Hz): 0.75 (1H, m, H-1,6), 1.02 (s), 1.05 (6H, s,
3
H-8,9), 1.42 (3H, s, H-10), 2.10 (2H, m, H-2), 2.55 (2H, m, H-5), 3.50 (1H, d, J = 7.55, H-4), 3.68 (2H, m, H-11), 5.40 (1H,
–1
+
m, H-12), 5.80 (2H, m, H-13). IR spectrum (ꢄ, cm ): 3640–3040 (OH), 1016 (S=O). GC–MS (m/z, I , %): 243 (1) [M + 1] ,
rel
152 (8), 135 (30), 119 (12), 109 (41), 93 (36), 81 (19), 71 (22), 67 (21), 55 (15).
(2,6,6-Trimethylbicyclo[3.1.1]hept-2-en-trans-4-ylthio)ethanoic Acid (29). PMR spectrum (ꢆ, ppm, J/Hz):
0.8 (s), 1.3 (6H, s, H-8,9), 1.35 (1H, d, J = 9.1, H -7), 1.62 (3H, dd, J
= 1.7, J
= 1.8, H-10), 2.0–2.4 (3H, m,
ꢁ
H-10 H-1
H-10 H-3
13
H-1,5, H -7), 3.1 (2H, m, SCH ), 3.45–3.51 (1H, m, H-4), 5.25–5.30 (1H, m, H-3), 11.1 (1H, s, COOH). C NMR spectrum
ꢀ
2
(ꢆ, ppm): 177.15 (C=O), 147.18 (C-2), 117.18 (C-3), 49.41 (SCH ), 48.50 (C-5), 47.20 (C-1), 46.15 (C-4), 33.30 (C-7), 29.30
2
(C-6), 27.51 (C-10), 23.80, 21.70 (C-8,9).
Methyl (2,6,6-trimetylbicyclo[3.1.1]hept-2-en-trans-4-ylthio)ethanoate (30). PMR spectrum (ꢆ, ppm, J/Hz):
0.79 (s), 1.21 (6H, s, H-8,9), 1.25 (1H, d, J = 9.6, H -7), 1.60 (3H, dd, J
= 1.7, J
= 1.8, H-10), 1.92 (1H, td,
ꢁ
H-10 H-1
H-10 H-3
J
= 1.2, J
= 5.5, J
= 10.4, H-1), 2.10–2.15 (1H, m, H-5), 2.18–2.21 (1H, m, J = 5.5, H -7), 3.14 (2H, m,
H-1 H-3
H-1 H-7ꢀ
H-1 H-7ꢁ
ꢀ
13
SCH ), 3.45-3.51 (1H, m, H-4), 3.60 (3H, s, OCH ), 5.22-5.26 (1H, m, H-3). C NMR spectrum (ꢆ, ppm): 170.05 (C=O),
2
3
150.10 (C-2), 118.15 (C-3), 53.25 (SCH ), 48.55 (C-5), 47.50 (C-1), 46.45 (C-4), 33.77 (C-7), 29.65 (C-6), 27.35 (C-10),
2
+
23.69, 21.65 (C-8,9). Mass spectrum (m/z, I , %): 240 (1) [M] , 167 (20), 147 (22), 134 (50), 119 (70), 105 (25), 93 (100),
rel
77 (55), 69 (68), 43 (98), 27 (31).
Methyl (6,6-dimethylbicyclo[3.1.1]hept-2-en-10-methylthio)ethanoate (33). PMR spectrum (ꢆ, ppm, J/Hz):
0.79 (s), 1.22 (6H, s, H-8,9), 1.13 (1H, m, H-7), 2.08 (1H, m, H-7), 2.16–2.25 (3H, m, H-4, H-5), 2.36, 2.41 (2H, J = 12,
AB
H-10, AB centers), 2.17 (1H, td, J
= 1.3, J
= 5.8, J
= 11.2, H-1), 3.15 (2H, m, SCH ), 3.48 (2H,
H-1 H-5
H-1 H-7ꢀ
H-1 H-7ꢁ
2
13
dd, J
= 2.7, J
= 5.7, H-4), 3.70 (3H, s, OCH ), 5.40–5.43 (1H, m, H-3).
C NMR spectrum (ꢆ, ppm):
H-4 H-5
H-4 H-3
3
170.15 (C=O), 150.30 (C-2), 118.15 (C-3), 53.05 (SCH ), 48.73 (C-5), 50.14 (C-1), 49.41 (C-4), 33.77 (C-7), 37.85 (C-6),
2
+
27.35 (C-10), 32.06, 21.32 (C-8,9). Mass spectrum (m/z, I , %): 240 (1) [M] , 197 (1), 167 (30), 147 (40), 135 (75), 119
rel
(100), 105 (50), 93 (80), 69 (90), 55 (35), 43 (95).
Starting monoterpenoids 1–3 and 17–24 and S-containing carane and pinane derivatives 4–16 and 25–33 were screened
for antifungal activty against mycelial and yeast-like fungi by a disk-diffusion application method on modified Sabureau agar.
Innoculations of test cultures (spore suspension) were calculated for 1 million CFU/dish. Cultures were incubated for 8 d at
28°C.
Test compounds were dissolved in volatile solvents (ethanol, acetone) and placed on paper disks calculated for 1 mg
of compound per disk. Disks were dried under sterile conditions for complete removal of solvent. The effectiveness of the
antifungal activity was estimated from the lysis zone (using the distance from the edge of the disk with the test compound to
the fungus growth zone) according to the usual method [16].
Strains preserved in the Kazan Institute of Epidemiology and Microbiology [A. niger BKM F-412, A. fumigatus
BKM F-219, P. tardum BKM F263, P. chrysogenum BKM F-347, C. albicans Y-4] and strains of yeast-like fungi and
dermatomycetes isolated from patient skin and mucous mycoses [C. albicans 228, C. parapsilosis, C. krusei, Rhodotorula
rubra, E. floccosum] [17, 18] were used for the biotests.
31

ACKNOWLEDGMENT
The work was supported financially by Grant RFBR 04-04-97511 ofi.
REFERENCES
1.
2.
3.
4.
5.
6.
K. A. Hammer, C. F. Carson, and T. V. Riley, J. Appl. Microbiol., 95, 853 (2003).
K. Nakahara, N. S. Alzoreky, and T. Yoshihashi, JARQ, 37, 249 (2003); http://www.jircas.affrc.go.jp.
J.-H. Lee, H.-Y. Yang, and H.-S. Lee, J. Microbiol. Biotechnol., 18, 497 (2008).
G. Stojanovic, I. Palic, and J. Ursic-Jankovic, Flavour Fragrance J., 21, 77 (2006).
B. F. Dababneh, J. Food Agric. Environ., 5, 158 (2007); http://www.world-food.net.
N. P. Artemova, G. Sh. Bikbulatova, I. A. Litvinov, O. N. Kataeva, and V. A. Naumov, Zh. Obshch. Khim., 59, 2718
(1989).
7.
8.
N. P. Artemova, G. Sh. Bikbulatova, V. V. Plemenkov, I. A. Litvinov, O. N. Kataeva, and L. N. Surkova,
Zh. Obshch. Khim., 60, 2374 (1990).
N. P. Artemova, G. Sh. Bikbulatova, V. V. Plemenkov, V. A. Naumov, and O. N. Kataeva, Khim. Prir. Soedin., 193
(1991).
9.
10.
11.
12.
N. P. Artemova, G. Sh. Bikbulatova, V. V. Plemenkov, and Yu. Ya. Efremov, Zh. Obshch. Khim., 61, 1481 (1991).
RF Pat. No. 2,296,749 (2007); Appl. 2005126295/04 (2005).
L. E. Nikitina, V. A. Startseva, S. A. Dieva, I. A. Vakulenko, and G. A. Shamov, Khim. Prir. Soedin., 146 (2006).
I. A. Vakulenko, V. A. Startseva, L. E. Nikitina, N. P. Artemova, L. L. Frolova, and A. V. Kuchin, Khim. Prir.
Soedin., 565 (2005).
13.
14.
A. Yu. Sergeev and Yu. V. Sergeev, Fungal Infections [in Russian], Binom, Moscow, 2003.
A. V. Kuchin, L. L. Frolova, I. V. Dreval , M. V. Panteleeva, E. U. Ipatova, and I. N. Alekseev, Izv. Ross. Akad.
Nauk, Khim., 413, 475 (2003).
15.
16.
17.
18.
A. Weissberger and E. S. Proskauer, Organic Solvents, 2nd Ed., rev. by J. A. Riddick and E. E. Toops, Jr.,
Interscience Pubs., Inc., New York, 1955.
N. B. Gradova, E. S. Babusenko, and I. B. Gornova, Laboratory Practicum in General Microbiology [in Russian],
DeLi Print, Moscow, 2004, pp. 111–113.
R. A. Araviiskii, N. N. Klimko, and N. V. Vasil eva, Diagnosis of Mycoses [in Russian], Izd. Dom SPbMAPO,
St. Petersburg, 2004, pp. 17–33.
D. A. Sutton, A. W. Fothergill, and M. G. Rinaldi, Guide to Clinically Significant Fungi, Williams & Wilkins,
Baltimore, 1997.
32