1,2,4-Triazolo mercapto and aminonitriles as potent antifungal agents

Article abstract of DOI:10.1016/S0960-894X(03)00378-0

A series of 3-mercapto-1,2,4-triazoles mono or disubstituted at 2-, 3- or 4-positions were synthesized and evaluated as antifungal agents. Many of these derivatives exhibit high activity against Candida albicans and Candida tropicalis.

Full text of DOI:10.1016/S0960-894X(03)00378-0

Bioorganic & Medicinal Chemistry Letters 13(2003) 2601–2605
1,2,4-Triazolo Mercapto and Aminonitriles as Potent
Antifungal Agents
Xavier Collin,a,* Armelle Sauleaua and Joel Coulonb
a
´
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, avenue du Pr Leon Bernard, F-35043 Rennes Cedex, France
bDepartment of Microbial Biochemistry, LCPME UMR7564 CNRS-UHP Nancy 1, Faculty of Pharmacy, 5 rue A. Lebrun,
F-54001 Nancy Cedex, France
Received 20 September 2002; revised 21 February 2003; accepted 4 April 2003
Abstract—A series of 3-mercapto-1,2,4-triazoles mono or disubstituted at 2-, 3- or 4-positions were synthesized and evaluated as
antifungal agents. Many of these derivatives exhibit high activity against Candida albicans and Candida tropicalis.
# 2003Elsevier Ltd. All rights reserved.
Introduction
tance to these drugs is rising, which clearly indicates an
urgent need for new antifungal agents.7
Triazoles and in particular 1,2,4-triazole nucleus have
been incorporated into a wide variety of therapeutically
interesting drug candidates including anti-inflammatory,
CNS stimulants, sedatives, antianxiety and antimicrobial
agents.1,2
To overcome rapid development of drug resistance, new
agents should preferably have chemical characteristics
that clearly differ from those of existing agents.
An ideal heterocyclic is the 4-amino-3-mercapto-4H-
1,2,4-triazole system which, by virtue of its vicinal
nucleophiles amino and mercapto groups constitues a
ready-made building block for construction of various
organic heterocycles.
Their antifungal activity is also documented.3,4 If
Amphotericin B is the most commonly drug used
against systemic mycoses despite its toxic effect on
humans, newer classes of antifungals including azole
derivatives (Fluconazole, an orally active triazole agent,
and Itraconazole), allylamines, thiocarbamates, fluoro-
pyrimidines are systemic antifungal agents actually
employed in patients with impaired immunity such as
those who have AIDS or are neutropenic as a result of
cancer therapy.
Prompted by the biological properties of 1,2,4-triazoles
and as a part of our general search concerning chemo-
therapeutically important azoles heterocycles,8 we deci-
ded to study new triazoles derivatives of 4-amino-3-
mercapto-1,2,4 triazoles. This work reported here
describes the synthesis and antifungal activity of some
mono substituted and disubstituted amino mercapto
triazoles. The influence of the thiol and cyano groups
was especially evaluated.
Invasive fungal infections constitute a major cause of
mortality for these patients. Candida albicans and
Cryptococcus neoformans are two of the most common
opportunistic fungi responsible for infections. For
Rabodinirina et al.5 and Hood et al.,6 the frequency of
deeply invasive candidiasis has increased 10-fold during
the past decade. Moreover, many infections due to
Candida spp are actually refractory to antifungal
therapy. While these new classes of compounds are now
frequently used in treatment of fungal infections, resis-
Chemistry
The 4-amino-3-mercapto-1,2,4 triazoles 1–4 (Fig. 1)
used in the present study for various nucleophilic sub-
stitution reactions, each bearing a different aromatic
unit attached in the 5-position, were obtained by
hydrazinolysis of the corresponding potassium-3-aroyl-
dithiocarbazates with excess hydrazine hydrate following
the Reid and Heindel procedure.9
*Corresponding author. Tel.: +33-2-2323-4843; fax: +33-2-2323-
4975; e-mail: xavier.collin@univ-rennes1.fr
0960-894X/03/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00378-0

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X. Collin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2601–2605
1 was heated under reflux in the presence of halonitriles
(or chloro-tetrazole) and Et3N (or K2CO3) in ethanol
(or acetone) until the triazole was consumed (Scheme 1);
the tautomeric 3-thiol/thione forms of 1 reacting
with halonitriles to the thiol form (3-S) or the poten-
tially tautomeric ring (2-NH), illustrating the alkylation
of ambident anions derived from aminomercapto
triazoles.8
Figure 1. 3-Thiol/thione forms of 4-amino-3-mercapto-1,2,4-triazoles.
We therefore required a functional group that could be
obtained in gram-scale amounts and could easily be
attached to the 3-mercapto or the 2-amino unit of 1. We
focused our attention on a cyano group at terminal
position of an aliphatic chain linked at compound 1 for
several reasons:
Cyano groups of compounds 8 and 10 were functiona-
lized into the corresponding amides (compounds 1116
and 1217) in the presence of sulfuric acid 36N.
The influence of 4-NH2 substituting groups such as a
pyrryl group (compounds 13 and 14, Scheme 2) or an
N-alkylnitrile group (compound 15) on the antifungal
activities was either investigated: 3-4-disubstituted 1,2,4-
triazoles 1318 and 1419 were prepared respectively from
products 5 and 8 using 2,5-dimethoxytetrahydrofurane
in glacial acetic acid (Clauson–Kass reaction). Com-
pound 2-4-disustituted 15 was previously synthesized
using a Michael addition.8
ꢀ Firstly, relatively little is known about antifungal
activity of triazolo nitriles.10
ꢀ Secondly, this work led to an extension of our
new chemicals methods of synthesis of triazolo-
nitriles discuted in a preceding publication.8 So
we are able to present here a series of amino or
mercapto-1,2,4-triazolonitriles for comparison of
their potent antifungal activity.
ꢀ Finally, the terminal cyano group could be easily
converted into larger aliphatic group (amide).
Chlorohydroxy or tetrazolo group which would
mimic the methylene nitrile susbtituent were also
introduced for comparisons.
The incorporation of an alternative chlorohydroxy
group in the 3-position of triazole 3 which would mimic
the methylene nitrile substituent, resulted of treating 3
with epichlorhydrin/NaHCO3 in absolute ethanol
(compound 16,20 Scheme 3).
Biological Results and Discussion
Various 3-mercaptonitriles (compounds 5,8 6,8 7,13 8,14
9,15 Scheme 1) or 2-aminonitriles (compound 10,8
Scheme 1) keeping a free 4-amino group (N–NH2), were
therefore synthesized using the following procedure:8
Antifungal activities of synthesized compounds were
determined by the disk diffusion method11 (Disk (1M
Whatman paper) diameter: 6 mm; Sabouraud solid
Scheme 1. Synthesis of 4-amino-3-mercapto-1,2,4-triazolonitriles S- or N-substituted. Reagents and conditions: (i) 1 (1 equiv), Cl–CH2–CN (4
equiv) or Cl–(CH2)3–CN (4 equiv) or Cl–CH(CH3)–CN (4 equiv) or Cl–CH2–C6H4–CN (1 equiv), Et3N, EtOH, reflux, 6 h; (ii) 1 (1 equiv), chloro-
tetrazole (1.2 equiv), K2CO3, acetone, reflux, 24 h; (iii) 1 (1 equiv), Cl–(CH2)2–CN (1.5 equiv), K2CO3, acetone, reflux, 24 h; (iv) 8 or 10, H2SO4
36 N, rt, 2 h.

X. Collin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2601–2605
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Scheme 2. Protection of 4-aminogroup of 4-amino-3-mercapto-1,2,4-triazolonitriles: (v) 5 or 8 (1 equiv), 2,5-dimethoxytetrahydrofuran (1 equiv),
CH3COOH, reflux, 1.5 h; (vi) 2 (1 equiv), acrylonitrile (1.5 equiv), K2CO3, CH3CN, rt, 7 h.
Scheme 3. (vii) 3 (1 equiv), NaHCO3 (1.17 equiv), epichlorhydrin (1.4 equiv), EtOH, rt, 3days.
medium (2% glucose, 1% peptone, 7% agar) was used
as growth medium) using following yeast strains: C.
albicans, C. tropicalis and Saccharomyces cerevisiae. The
compounds were dissolved in dimethylformamide at the
concentration of 40 mg/mL (excepted Amphotericin B,
20 mg/mL). The results are summarized in Table 1.
that of 1, we focused our comments on results obtained
on C. albicans and C. tropicalis yeasts.
Modulation of the phenyl cycle
Introduction of a chlorine atom in the para position
(3) and incorporation of a methylene chain between
the phenyl and the 1,2,4-triazole cycle (2) increased the
antifungal activity from 30 to 50% on the yeasts spe-
cies, affording the most efficient compounds of the
series.
All activities were referred to 5-phenyl-4-amino-3-mer-
capto-1,2,4-triazole 1. Taking into account that phar-
macomodulations effected did not involve any increase
of antifungal activity against S. cerevisiae compared to
Table 1. Antifungal activities of synthesized 1,2,4-triazoles

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X. Collin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2601–2605
The presence of a p-methyl group (compound 4) did not
exert any significant modification on the antifungal
activity of original compound 1.
electron-releasing group, the activity would tend to be
maximum (compounds 2 and 3). However, this conclusion
calls for a deeper study of electron density calculations.
The second observation that is worth noting was that
keeping the aromatic group constant, attachment of an
alkylnitrile group to the thiol function enhanced the
antifungal activity (5, 6 and 7), perhaps due to the sig-
nificant interaction of CN function (electron-with-
drawing group) with free amino group at N-4 (electron-
releasing groups). It would be confirmed by the
diminution of antifungal activity observed in the fol-
lowing cases, cramping or suppressing such a dipole–
dipole interaction:
Substitution on the sulphur atome
Keeping the aromatic group constant on the tetrazolyl
carbon 5 (phenyl group), alkylation of the thiol function
by a methylcyano group (5) led to an increase (1 mm) of
the diameter of inhibition on each yeast compared to
that of 1.
Enhancement of the alkyl chain (6) and introduction of
a phenyl group (8) between cyano group and sulfur
atom did not increase the antifungal activity. On the
other hand, introduction of a methyl ramification lead-
ing to the presence of an asymmetrical carbone atome
on this aliphatic chain (7) induced the most significant
diameters of inhibition in this serie (12 mm on each
yeast).
ꢀ important steric overcrowding due to a benzyl
group (8);
ꢀ protection of the amino function (13 and 14);
ꢀ replacement of the nitrile group by an amidic
function (11 and 12), a tetrazole cycle (9) or a 4-
chloro-3-hydroxybutyl group (16);
Substitution of the thiol function by a tetrazolo cycle (9)
significantly decreased the activity.
ꢀ attachment of the nitrile group at nitrogen-2
leading to steric removing of this group from the
amino function at C-4 (10 and 15 where protection
of amino group is also effective).
Transformation of the nitrile group of compound 8 into
amidic function (11) was either out of interest for anti-
fungal activity.
Moreover, it is partly in contradiction with the purpose
of Ghannoum and coll. considering that both NH2 and
SH groups should be free for antimicrobial activity.3
According to our studies, if a free amino group is
necessary for optimal activity, implication of the thiol
group in a thioether function leading to the introduction
of a cyanoalkyl group increases the antifungal activity.
At last, introduction of a 4-chloro-3-hydroxybutyl
group on the sulfur atom of 3 to form compound 16 was
not favorable.
Protection of the NH2 group
Protection of the NH2 function of compounds 5 and 8
by a pyrole group (13 and 14) did not favour the anti-
fungal activity. It is in harmony with the studies of
Beuchet and coll. who suggested that presence of a pri-
mary amine was necessary for the expression of the
antifungal activity of amino sterols.12
In continuation of our researches, further extensive
studies for the synthesis of new 1,2,4-triazoles (dimer
structures) which may provide useful antifungal agents
to combat infections such as candidiasis are now in
progress.
Substitution on the intracyclic 2-nitrogen atom
We examined the interest of the tautomeric thiol/thione
form illustrated by the alkylation of ambident anions
derived from 1. Substitution of the hydrogen of the
intracyclic 2-nitrogen atom by an ethylcyano group
(10), leading to the chemical stabilization of the tauto-
meric thione form, decreased the inhibitory activity
against yeasts studied, compared to these of compound
1 and S-substituted 5 and 6 (thiol form) analogues.
Supplementary substitution of the amino group in
nitrogen 4 by the same ethylcyano group (15) did not
improve antifungal activity.
References and Notes
1. Heindel, N. D.; Reid, J. R. J. Heterocycl. Chem. 1980, 17,
1087.
2. Holla, B. S.; Kalluraya, B.; Sridhar, K. R.; Drake, E.;
Thomas, L. M.; Bhandary, K. K.; Levine, M. J. Eur. J. Med.
Chem. 1994, 29, 301.
3. Ghannoum, M. A.; Eweiss, N. F.; Bahajaj, A. A.; Qureshi,
M. A. Microbios. 1983, 37, 151.
4. Demirayak, S.; Benkli, K.; Guven, K. Eur. J. Med. Chem.
2000, 35, 1037.
5. Rabodinirina, M.; Piens, M. A.; Monier, M. F.; Gueho, E.;
Fiere, D.; Mojon, M. Eur. J. Clin. Microbiol. Infect. Dis. 1994,
13, 152.
Conclusion
6. Hood, S.; Denning, D. W. J. Antimicrob. Chemother. 1996,
37, 71.
In conclusion, the structure–antifungal activity rela-
tionships may be interpreted as follows: the balance of
overhall electron density present on both the aromatic
and heterocyclic rings seems to be responsible for the
activity. It would be confirmed by the fact that with an
7. Alexander, B. D. Perfect Drugs. 1997, 54, 657.
8. Sauleau, A.; Sauleau, J. Phosphorus, Sulfur Silicon 2000,
167, 219.
9. Reid, J. R.; Heindel, N. D. J. Heterocycl. Chem. 1976, 13,
925.

X. Collin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2601–2605
2605
10. Martin, M. V. J. Antimicrob. Chemother. 1999, 44, 429.
11. Robert-Dernuet, S. Antibiotiques et Antibiogrammes 1995,
322.
16. Physical data of synthesized compound 11 (Scheme 1): IR
(n cmꢁ1, KBr): 3402 (NH2 amide), 3350 and 3210 (NH2
amine), 1658 (CO). 1H NMR (d, 500 MHz, DMSO-d6): 4.50 (s,
2H, CH2); 6.10 (s, 2H, NH2 amine); 7.30 (s, 1H, NH amide);
7.50 (m, 5H, m- and p-Ph, o-Ph–CH2); 7.80 (m, 2H, m-Ph–
CH2); 7.90 (s, 1H, NH amide); 8.00 (m, 2H, o-Ph).
12. Beuchet, P.; Dherbomez, M.; Elkiel, G.; Charles, G.;
Letourneux, Y. Bioorg. Med. Chem. Lett. 1999, 9, 1599.
13. Physical data of synthesized compound 7 (Scheme 1): IR
(n cmꢁ1, KBr): 3342 and 3104 (NH2), 2240 (CN). 1H NMR (d,
500 MHz, DMSO-d6): 1.70 (d, J=7 Hz, 3H, CH3); 4.80 (q,
J=7 Hz, 1H, CH); 6.25 (s, 2H, NH2); 7.55 (m, 3H, m- and p-
Ph); 8.00 (m, 2H, o-Ph). 13C NMR (d, 500 MHz, DMSO-d6):
8.2 (q, J=33 Hz, CH3); 27.8 (d, J=54 Hz, CH); 120.1, (m,
CN); 126.4 (t, J=7 Hz i-Ph); 127.8 (dt, J=162 Hz, 6.5 Hz, o-
Ph); 128.5 (dd, J=160 Hz, 7 Hz, m-Ph); 129.8 (dt, J=161 Hz,
7 Hz, p-Ph); 150.3(d, J=3Hz, C-Ph); 154.4 (s, C–S).
17. Physical data of synthesized compound 12 (Scheme 1): IR
(n cmꢁ1, KBr): 3414 (NH2 amide), 3292 and 3192 (NH2
amine), 1668 (CO). 1H NMR (d, 500 MHz, DMSO-d6): 2.65 (t,
J=7.5 Hz), 2H, CH2–CO); 4.35 (t, J=7.5 Hz, 2H, CH2–N);
5.85 (s, 2H NH2); 6.95 (s, 1H NH amide); 7.45 (s, 1H, NH
amide); 7.55 (m, 3H, m- and p-Ph); 8.00 (m, 2H, o-Ph).
18. Physical data of synthesized compound 13 (Scheme 2): IR
(n cmꢁ1, KBr): 2250 (CN). 1H NMR (d, 500 MHz, DMSO-d6):
4.30 (s, 2H, CH2); 6.35 (t, J=2.2 Hz, 2H, bb0 pyrrole); 7.30 (t,
J=2.2 Hz), 2H, aa0 pyrrole); 7.35 (d, 8 Hz, 2H, o-Ph); 7.40 (t,
J=7.8 Hz, 2H, m-Ph); 7.50 (t, J=7.5 Hz, 1H, p-Ph). 13C
NMR (d, 500 MHz, DMSO-d6): 17.7 (t, J=152 Hz, CH2);
109.6 (dm, J=174 Hz and 4 Hz, bb0 pyrrole); 117.1 (t, J=8 Hz,
CN); 122.0 (dm, J=194 Hz and 4 Hz, aa0 pyrrole); 124.5 (t,
J=8.5 Hz, i-Ph); 126.2 (dt, J=162 Hz and 7 Hz, o-Ph); 128.9
(dd, J=163Hz and 7 Hz, m-Ph); 130.8 (dt, J=163Hz and 7 Hz,
p-Ph); 150.2 (t, J=5.5 Hz, C–Ph); 153.4 (t, J=3.6 Hz, C–S).
19. Physical data of synthesized compound 14 (Scheme 2): IR
(n cmꢁ1, KBr): 2224 (CN). 1H NMR (d, 500 MHz, DMSO-d6):
4.50 (s, 2H, CH2); 6.30 (t, J=2.2 Hz, 2H bb0 pyrrole); 7.10 (t,
J=2.2 Hz, 2H, aa0 pyrrole); 7.30 (d, J=8.4 Hz, 2H, o-Ph);
7.40 (t, J=7.6 Hz, 2H, m-Ph); 7.45 (t, J=7.5 Hz, 1H, p-Ph);
7.65 (d, J=8.3Hz, 2H, o-Ph-CH2); 7.80 (d, J=8.3Hz, 2H, m-
Ph–CH2).
14. Physical data of synthesized compound 8 (Scheme 1): IR
(n cmꢁ1, KBr): 3342 and 3174 (NH2), 2226 (CN). 1H NMR (d,
500 MHz, DMSO-d6): 4.50 (s, 2H, CH2); 6.10 (s, 2H, NH2);
7.50 (m, 3H m- and p-Ph); 7.65 (m, 2H, o-Ph–CH2); 7.80 (m,
2H, m-Ph–CH2); 8.00 (m, 2H, o-Ph). 13C NMR (d, 500 MHz,
DMSO-d6): 34.1 (t, J=147 Hz, CH2); 109.9 (t, J=8 Hz, C-
CN); 118.7 (m, CN); 126.7 (t, J=7 Hz, i-Ph); 127.7 (dt, J=163
Hz and 5 Hz, o-Ph);128.4 (dd, J=161 Hz and 7 Hz, m-Ph);
129.6 (dt, J=161 Hz and 7 Hz, p-Ph); 129.9 (dd, J=165 Hz
and 5 Hz, m-Ph–CN); 132.2 (dd, J=168 Hz and 6 Hz, o-Ph-
CN); 143.8 (m, p-Ph–CN); 152.7 (t, C–Ph);154.2, (s, C–S).
15. Physical data of synthesized compound 9 (Scheme 1): IR
1
(n cmꢁ1, KBr): 3328 and 3182 (NH2). H NMR (d, 500 MHz,
DMSO-d6): 6.30 (s, 2H, NH2); 7.55 (m, 3H m- and p-Ph); 7.70,
7.80, 8.00 (m, 6H, Ph). 13C NMR (d, 500 MHz, DMSO-d6):
125.0 (dd, J=166 Hz and 6 Hz); 126.1 (t, i-Ph); 127.9 (d,
J=163Hz, o-Ph); 128.5 (dd, 161 Hz and 6 Hz, m-Ph); 129.8
(dd, J=165 Hz and 7 Hz); 130.1 (dt, J=162 Hz and 8 Hz, p-
Ph); 130.9 (dt, J=162 Hz and 8 Hz, p-Ph tetrazole); 132.9 (t,
J=7 Hz, i-Ph tetrazole); 146.3(s C–Ph); 149.7 (s, C–S); 154.8
(s, S–C(¼N)–N).
20. Physical data of synthesized compound 16 (Scheme 3): 1H
NMR (d, 500 MHz, DMSO-d6): 2.50 (s, 3H, CH3; 3.30 (AB,
2H, CH2–S); 3.69 (AB, 2H, CH2–Cl); 4.03(m, 1H, CH–O);
5.73(s, 1H, OH); 6.15 (s, 2H, NH ); 7.60 (d, 2H, m-Ph); 8.04
2
(d, 2H, o-Ph).