The Triazole Ring as a Privileged Scaffold for Putative Antifungals: Synthesis and Evaluation of a Series of New Analogues

Article abstract of DOI:10.1002/cmdc.202000312

The significant antifungal activity of a series of novel 1,2,4-triazole derivatives against different strains of Candida albicans, Candida krusei and Aspergillus fumigatus, compared to the commercial fungicides ketoconazole and itraconazole, is reported. Systemic mycosis and invasive fungal infections, whether from immunodeficiency or hospital-acquired infection, have been on an upward trend for several years. The 1,2,4-triazole ring substituted with other aromatic and heteroaromatic systems plays an important role in the field of antifungal drug discovery and development. Thus, an extensive series of 29 triazoles, substituted in different positions with a variety of aromatic rings, has been designed, synthesized, and evaluated for their fungicidal activity. Almost all the agents tested in vitro showed high activity against all examined fungal strains. It is noteworthy that, in the case of A. fumigatus, all the examined compounds achieved equal or higher antifungal activity than ketoconazole, but less activity than itraconazole. Among all the derivatives studied, the dichlorourea analogue and bromo-substituted triazole stand out as the most promising compounds. Quantitative structure-activity relationship (QSAR) models were built for a systematic structure-activity relationship (SAR) profile to explain and potentially explore the potency characteristics of 1,2,4-triazole analogues.

Full text of DOI:10.1002/cmdc.202000312

Accepted Article
Title: The Triazole ring as a Privileged Scaffold for Putative Antifungals:
Synthesis and Evaluation of a Series of New analogues
Authors: Grigoris Zoidis, Eftichia Kritsi, Paulina Lecinska, Marija
Ivanov, Panagiotis Zoumpoulakis, Marina Sokovic, and Marco
Catto
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000312
Link to VoR: https://doi.org/10.1002/cmdc.202000312
01/2020

10.1002/cmdc.202000312
ChemMedChem
FULL PAPER
The Triazole ring as a Privileged Scaffold for Putative Antifungals:
Synthesis and Evaluation of a Series of New analogues
Grigoris Zoidis,*[a] Eftichia Kritsi,[b] Paulina Lecinska,[c] Marija Ivanov,[d] Panagiotis Zoumpoulakis,[b]
Marina Sokovic,[d] and Marco Catto[c]
This paper is dedicated to Professor Angelo Carotti on the occasion of his 74th birthday
[a]
Prof. Dr. G. Zoidis (https://orcid.org/0000-0002-9442-5186)
School of Health Sciences, Department of Pharmacy, Division of Pharmaceutical Chemistry
National and Kapodistrian University of Athens
Panepistimiopolis-Zografou, GR-15771, Athens (Greece)
E-mail: zoidis@pharm.uoa.gr
[b]
[c]
[d]
Dr. E. Kritsi (https://orcid.org/0000-0002-3975-0832), Dr. P. Zoumpoulakis (https://orcid.org/0000-0001-9348-6078)
Institute of Chemical Biology, National Hellenic Research Foundation,
Vas. Constantinou Ave. 48, 11635 Athens (Greece)
Dr. P. Lecinska, Dr. M. Catto (https://orcid.org/0000-0002-8411-304X)
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di
Bari “Aldo Moro”, via E. Orabona 4, 70125 Bari (Italy)
Dr. Marija Ivanov (https://orcid.org/0000-0002-2480-5490), Dr. M. Sokovic (https://orcid.org/0000-0002-7381-756X)
Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”-National Institute of Republic of Serbia,
University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade (Serbia)
Supporting information for this article is given via a link at the end of the document.
usually life-threatening human infections.[6-7] Candida albicans, a
common member of the normal human microbiome, constitutes
the most frequent cause of severe systemic fungal infections in
immunocompromised patients and it is classified as the fourth
most common cause of hospitalized bloodstream infections.[8-9]
Epidemiological results indicated that Candida albicans is
associated with > 400,000 cases per year of systemic candidiasis
in a global level and its mortality rate ranges from 46% to 75%.[5]
Additionally, invasive candidiasis has mortality rates 40 to 60%,
and are most commonly caused by C. albicans, C. glabrata, C.
tropicalis, C. parapsilosis, and C. krusei. The latter is an emerging
nosocomial pathogen, presenting a significant increase in the last
decade.[10] Invasive aspergillosis, the disease caused mainly by
Aspergillus fumigatus, has been estimated to induce over
200,000 cases in immunosuppressive hosts and the prevalence
of mortality ranging from 30% to 95%.[5,11] Also, Aspergillus
fumigatus is responsible for chronic pulmonary aspergillosis,
affecting more than 3 million people worldwide.[5,12]
Abstract: The significant antifungal activity of a series of novel 1,2,4-
triazole derivatives against different strains of Candida albicans,
Candida krusei and Aspergillus fumigatus, compared to the
commercial fungicide ketoconazole and itraconazole, is reported.
Systemic mycosis and invasive fungal infections, whether from
immunodeficiency or hospital-acquired infection, have been on an
upward trend for several years. The 1,2,4-triazole ring substituted with
other aromatic and heteroaromatic systems plays an important role in
the field of antifungal drug discovery and development. Thus, an
extensive series of 29 triazoles, substituted in different positions with
a
variety of aromatic rings, were designed, synthesized, and
evaluated for their fungicidal activity. Almost all the in vitro tested
agents showed a high activity against all examined fungal strains. It is
noteworthy, that in the case of Aspergillus fumigatus all the examined
compounds achieved equal or higher antifungal activity than
ketoconazole, but less activity than itraconazole. Among all the
derivatives studied, the dichloro urea analogue and bromo substituted
triazole, stand out as the most promising compounds. Quantitative
structure activity relationship (QSAR) models were built for a
systematic structure activity relationship (SAR) profile to explain and
potentially explore the potency characteristics of 1,2,4-triazole
analogs.
Despite the urgent need for effective confrontation of invasive
mycoses, currently available antifungal pipeline includes only four
major drug classes, polyenes, flucytosine, azoles and
echinocandins, targeting three specific fungal metabolic pathways
(the ergosterol biosynthesis, the fungal cell wall and the fungal
DNA/RNA).[13] The number of antifungal drugs is restricted
because of their limited clinical efficacy, narrow spectrum of
fungal strains, adverse pharmacokinetic profiles, severe side
effects, and interactions with other drugs.[14-15] The increasing
antimicrobial drug resistance from the emerging fungal pathogens
provides also a challenging task to be faced,[16-17] thus the
discovery of novel therapeutic agents is of paramount importance.
Since their discovery, azoles have gained widespread attention,
exhibiting the most widely used category of antifungal agents in
clinical practice.[18] Their antifungal mode of action is based on the
Introduction
In recent years, the prevalence of invasive fungal infections (IFIs)
has been raised due to the increasing number of
immunosuppressive population, the high frequency of bone
marrow and organ transplants, the use of antineoplastic agents,
the excessive use of antibiotics and the prolonged use of
corticosteroids.[1-3] The contribution of invasive fungal infections to
human morbidity and mortality is highlighted by the fact that they
are responsible for over one million deaths per year.[4-5] Candida
albicans, Aspergillus fumigatus, and Cryptococcus neoformans
are the most common fungal pathogens, causing serious and
1
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10.1002/cmdc.202000312
ChemMedChem
FULL PAPER
inhibition of the cytochrome P450 dependent lanosterol 14α
triazoles remains still in the frontline of the pharmaceutical
research for novel antifungals.[26] In line with this, the present
study attempts to discover novel triazoles with putative antifungal
activity. For this scope, a series of 29 triazole derivatives (Table
1) were synthesized and their activity was evaluated against three
different pathogenic fungal strains namely Candida albicans,
Candida krusei and Aspergillus fumigatus. In order to provide
greater insight into the chemical features that may enhance
antifungal activity, the polar triazole ring was substituted with a
variety of chemically diverse but pharmacologically interesting
groups in different positions of the heterocycle. Towards this
direction, urea and amide groups were utilized as linkers and
different electron withdrawing (EWG: -Cl, -Br, -F) and electron
donating groups (EDG: -NH2, -OCH3) were added on the aromatic
rings. Quantitative structure activity relationship models (QSAR)
were generated in an effort to provide a putative explanation of
the correlation among key parameters of the synthesized
compounds that may influence their antifungal activity.
demethylase enzyme (CYP51A),
a key enzyme in the
biosynthesis of ergosterol which is a significant component of the
fungal cell membrane. Particularly, a lone pair of electrons on an
unhindered nitrogen atom of azoles binds to the heme iron atom,
located in the active site of the enzyme CYP51 (encoded by
ERG11 gene), inducing the azoles mechanism of action.[19-21]
Azole drugs are classified into two groups bearing characteristic
(a) imidazole and (b) triazole rings. The firstly developed
imidazole drugs, such as miconazole, clotrimazole and
ketoconazole, exhibited high toxicity, unfavorable side effects and
a plethora of interactions with other drugs. Triazoles overcome
several limitations, displaying lower toxicity and stronger
antifungal activity compared to imidazoles and other
antifungals.[22] Recent evidences have proved that new
generation triazoles,[23] such as voriconazole and posaconazole
possess potent activity against Candida and Aspergillus
species.[24] In 2015 isavuconazole was approved for the treatment
of invasive aspergillosis, indicating the pivotal role of triazoles in
the fungus treatment.[25] Consequently, the identification of novel
1
2 R1=-CH2C6H5, R2=-C6H5
11 R3=-C6H5, R4=H
3 R1=-C6H5, R2=-CH2C6H5
12 R3=-C6H3(Cl)-2,(Cl)-4, R4=H
13 R3=-C6H4(Cl)-3, R4=H
14 R3=-CH2C6H5, R4=H
15 R3=-CH2C10H7, R4=H
4 R1=-CH2C6H4(CH3O)-4, R2=-C6H5
5 R1=-CH2C6H4(Br)-4, R2=-C6H5
6 R1=-CH2C6H3(Cl)-3,(Cl)-4, R2=-C6H5
7 R1=-CH2C6H3(Cl)-2,(F)-6, R2=-C6H4(Cl)-4
8 R1=-CH2C6H4(CH3O)-4, R2=-C6H4(Cl)-4
9 R1=-CH2C6H4(ΝΗ2)-4, R2=-C6H4(Cl)-4
10 R1=-CH2C6H4(C6H5)-4, R2=-C6H4(Cl)-4
30 R1=-CH2C6H4(NH2)-4, R2=-C6H5
16 R3=-C6H5, R4=Cl
17 R3=-CH2CH2C6H5, R4=H
18 R3=-C6H11, R4=H
31 R1=-CH2C6H4(ΝΗ2)-3, R2=-C6H4(Cl)-3
32 R1=-CH2C6H4(ΝΗ2)-3, R2=-C6H4(Cl)-4
19 R5=(Cl)-4
20 R5=(Cl)-3
21 R6=H, R7=H
25 R8=-C6H5, R9=-CH2C6H5
26 R8=-C6H5, R9=-C4H3S
27 R8=-C6H5, R9=-C5H4N
28 R8=-C7H5O2, R9=-C6H4(Cl)-4
29 R8=-C6H4[-NHSO2C6H4(CH4)-4]-4, R9=-C6H5
22 R6=(Cl)-4, R7=H
23 R6=(Cl)-3, R7=(Cl)-3
24 R6=(Cl)-3, R7=(Cl)-4
Table 1. The chemical structures of the synthesized triazole derivatives
Results and Discussion
Scheme 1. Reagents and conditions: (a) sodium ascorbate, CuSO4·5H2O, t-
BuOH/H2O=1/1, rt, 16 h, (85%).
Chemistry
In order to synthesize the 1-benzyl-4-phenyl-1H-1,2,3-triazole 1,
[27] we performed a Cu(I)-catalyzed [3+2]-dipolar cycloaddition of
benzyl azide 33 and benzonitrile 34 (Scheme 1).[28]
The synthesis of 3-aryl-5-benzyl-1H-1,2,4-triazole scaffold was
achieved through a straightforward click procedure, starting from
commercial
benzhydrazides
35
and
substituted
phenylacetonitriles 36. Dibenzyl derivative 25 and compound 3
were prepared with the same procedure, from 2-
phenylacetohydrazide and phenylacetonitrile the first and 2-
phenylacetohydrazide with benzonitrile the latter. The reaction
2
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10.1002/cmdc.202000312
ChemMedChem
FULL PAPER
yields and times were successfully optimized by using microwave
heating (Scheme 2). Specifically, in the case of compounds 2 and
3 the reaction times were reduced to 2 hours and the yields were
considerable improved.[29]
Antifungal activity evaluation
For the identification of antifungal activity of the synthesized
triazole derivatives, in vitro assays were performed against
different strains of Candida albicans (strains 475/15, 10/15 and
ATCC10231), Candida krusei (strain H1/16) and Aspergillus
fumigatus (strain ATCC9197). The minimal inhibitory
concentrations (MIC values) and the minimal fungicidal
concentrations (MFC values) of the tested compounds were
detected in comparison to ketoconazole (an imidazole) and
itraconazole (a triazole), the azole drugs used as positive control
(Table 2).
Compounds
2 and 3
Ar
R
H
Yields (%)
85 and 60
From the in vitro results, it is obvious that all synthetic derivatives
displayed significant antifungal activity against all examined
fungal strains. The comparison of the antifungal activity against
the different strains of Candida albicans proved that all
compounds present equivalent activity and, in some cases
(compounds 2, 5, 12, 17, 18, 22, 26, 29) Candida albicans
ATCC10231 characterized as the most sensitive strain. Against
the latter strain the amide analogues (11-20) proved to be more
potent than the less bulky triazoles (1-9 and 25-28) and slightly
more potent than the urea counterparts (11 vs 21), with the
exception of compound 24. As far as the substitution of the less
bulky triazoles (1-9) is concerned, the amino group in compound
9 (hydrogen bond donor) seems to play a pivotal role, probably
via hydrogen bonding interaction. Additionally, the observed MIC
(0.288-3.369 μmol·mL-1) and MFC (0.575-6.738 μmol·mL-1)
values of the tested triazoles against Candida krusei H1/16 are
equal or higher compared to Candida albicans strains, with six
exceptions (compounds 1, 3, 4, 9, 16 and 29), rendering it as the
most resistant species for the studied class of compounds, for
which the same SAR trend was generally observed.
4
5
6
7
8
4-OCH3
4-Br
83
89
85
65
55
50
59
55
37
64
phenyl
3,4-diCl
2-F-6-Cl
4-OCH3
4-NH2
4-Ph
9
4-chlorophenyl
10
28
26
27
3,4-(OCH2O)
H
thien-2-yl
pyrid-3-yl
H
Scheme 2. Reagents and conditions: (a) K2CO3, n-BuOH, MW, 150 °C, 3-4 h
(TLC monitoring), 40-90 %.
Amides 11-20 (Scheme 3) and ureas 21-24 (Scheme 4) were
obtained from aniline derivatives 9 and 30-32, by the reaction with
commercial acyl chlorides, carboxylic acids (Scheme 3) and
isocyanates (Scheme 4), respectively. Sulfonamide 29 was
prepared from benzenesulfonyl chloride according to the
conditions shown in Scheme 3.
It is noteworthy that, in the case of Aspergillus fumigatus
ATCC9197 all the examined compounds achieved equal or higher
antifungal activity than ketoconazole (MIC=0.433 μmol·mL-1 and
MFC=1.261 μmol·mL-1) apart from compounds 18 (MIC=0.860
μmol·mL-1 and MFC=1.720 μmol·mL-1) and 29 (MIC=0.922
μmol·mL-1 and MFC=1.818 μmol·mL-1) (Table 2). However, they
were less active than itraconazole. Compound 24 possessed the
highest potency. Compounds 24, 5, 4, and 6 were the most potent
against Aspergillus fumigatus, with MIC values ranged from 0.114
to 0.230 μmol·mL-1 and MFC values among 0.228-0.920
μmol·mL-1 (Table 2). Interestingly, against Aspergillus fumigatus
amide analogues (11-20), appear to follow a similar trend that was
observed for the other species, with similar potencies as the urea
counterparts (11 vs 21). The effect of the substitution position on
ring C on fungicidal activity was also investigated. It seems that
substitution on ring C did not seem to play a pivotal role on
potency. Finally, replacement of the aromatic ring B with a
heterocycle (thiophene, pyridine) (26 or 27 vs 2) led to a 2.2 and
1.5-fold decrease in potency respectively. Interestingly, tautomers
2 and 3 present different activities against Candida albicans
Compounds
R
R΄
Ph
Subst. position
11
12
13
14
15
16
17
18
19
20
4
4
4
4
4
4
4
4
3
3
2,4-diClPh
3-ClPh
CH2Ph
CH2Naphth-2-yl
Ph
H
4-Cl
H
(CH2)2Ph
CyHexyl
Ph
4-Cl
3-Cl
Ph
Scheme 3. Reagents and conditions: (a) DIPEA (3eq), dry THF, 0 ° to rt, 3-5 h
(TLC monitoring), 29-92 %; (b) For compounds 13-15 and 17: N-Methyl
morpholine (NMM), HOBt, THF, EDC, 0 °C to rt, under argon, (TLC monitoring)
16 h, 44-72 %.
Compounds
R
H
H
3-Cl
4-Cl
R΄
H
4-Cl
3-Cl
3-Cl
Subst. position
21
22
23
24
4
4
3
3
ATCC10231 and Aspergillus fumigatus with compound
2
presenting higher antifungal activity in both strains.
Scheme 4. Reagents and conditions: (a) Dry THF, 0 °C to rt, up to 48 h (TLC
monitoring), 26-80 %.
3
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10.1002/cmdc.202000312
ChemMedChem
FULL PAPER
Table 2. Minimal inhibitory (MIC) and fungicidal (MFC) concentration values of the synthesized compounds (µmol·mL−1).
Compounds
Candida albicans
475/15
Candida albicans
10/15
Candida albicans
ATCC10231
Candida krusei
Aspergillus fumigatus
ATCC9197
H1/16
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
1
2
0.637±0.004
1.062±0.01
1.062±0.01
1.696±0.02
1.528±0.01
0.789±0.004
0.869±0.004
0.801±0.004
0.562±0.004
0.549±0.005
0.508±0.008
0.992±0.02
0.463±0.002
0.488±0.002
0.645±0.003
0.566±0.005
0.497±0.004
0.721±0.004
1.275±0.01
2.125±0.02
2.125±0.01
3.392±0.02
3.024±0.02
1.578±0.02
1.738±0.02
1.601±0.02
1.124±0.015
1.041±0.01
1.016±0.01
1.984±0.03
0.926±0.01
0.977±0.01
1.290±0.01
1.131±0.01
0.993±0.01
1.443±0.01
0.637±0.004
1.062±0.01
1.062±0.01
1.696±0.02
1.528±0.01
0.789±0.004
0.869±0.004
0.801±0.005
0.562±0.005
0.549±0.005
0.508±0.008
0.992±0.02
0.463±0.004
0.488±0.004
0.645±0.004
0.566±0.005
0.497±0.004
0.721±0.004
1.275±0.01
2.125±0.02
2.125±0.02
3.392±0.02
3.024±0.02
1.578±0.02
1.738±0.02
1.601±0.01
1.124±0.01
1.041±0.01
1.016±0.01
1.984±0.02
0.926±0.01
0.977±0.01
1.290±0.01
1.131±0.01
0.993±0.01
1.443±0.01
0.637±0.004
0.510±0.004
1.062±0.02
0.867±0.01
0.764±0.004
0.789±0.004
0.869±0.004
0.801±0.005
0.562±0.004
1.014±0.01
0.508±0.008
0.496±0.01
0.463±0.004
0.488±0.004
0.645±0.004
0.566±0.005
0.235±0.002
0.361±0.002
1.275±0.01
2.125±0.02
2.125±0.03
1.696±0.02
1.528±0.02
1.578±0.02
1.738±0.02
1.601±0.02
1.124±0.02
2.082±0.02
1.016±0.01
0.992±0.02
0.926±0.02
0.977±0.01
1.290±0.01
1.131±0.01
0.497±0.006
0.721±0.01
0.297±0.001
0.510±0.004
0.510±0.004
0.867±0.01
1.528±0.02
0.953±0.01
2.080±0.02
1.935±0.02
0.316±0.004
2.573±0.02
1.129±0.01
0.590±0.004
0.540±0.004
0.597±0.004
2.867±0.04
0.334±0.004
0.288±0.002
0.444±0.002
0.637±0.004
2.125±0.02
1.062±0.01
1.696±0.03
3.024±0.02
1.910±0.02
4.159±0.05
3.903±0.05
0.632±0.01
5.118±0.04
2.257±0.02
1.181±0.02
1.080±0.02
1.194±0.01
5.735±0.06
0.669±0.01
0.575±0.004
0.888±0.01
0.382±0.004
0.340±0.004
0.637±0.004
0.226±0.001
0.223±0.001
0.230±0.001
0.497±0.005
0.467±0.005
0.632±0.005
0.636±0.005
0.282±0.004
0.283±0.001
0.257±0.001
0.298±0.002
0.765±0.004
0.643±0.005
0.288±0.002
0.860±0.01
0.765±0.004
1.232±0.01
1.275±0.01
0.980±0.01
0.891±0.004
0.920±0.01
0.993±0.01
0.934±0.01
1.264±0.01
1.272±0.01
0.564±0.008
0.567±0.004
0.514±0.004
0.597±0.003
1.529±0.02
1.286±0.01
0.575±0.004
1.720±0.015
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
4
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10.1002/cmdc.202000312
ChemMedChem
FULL PAPER
19
0.746±0.004
1.440±0.01
1.517±0.02
0.746±0.004
1.440±0.01
0.568±0.008
1.040±0.01
0.730±0.004
0.182±0.002
1.043±0.01
1.285±0.01
0.889±0.02
0.733±0.004
1.562±0.01
0.013±0.0001
0.013±0.0002
1.517±0.01
2.880±0.02
1.137±0.01
2.080±0.02
1.460±0.01
0.365±0.004
2.086±0.02
2.569±0.02
1.777±0.02
1.466±0.01
3.124±0.02
0.094±0.0002
0.026±0.0004
0.746±0.004
1.440±0.02
1.517±0.01
2.880±0.02
1.137±0.01
1.040±0.01
1.460±0.01
0.365±0.004
2.086±0.02
1.285±0.01
1.777±0.02
1.466±0.02
1.562±0.02
0.012±0.0001
0.052±0.0004
0.900±0.01
1.800±0.02
6.738±0.04
2.761±0.04
2.476±0.02
3.468±0.02
0.912±0.01
2.487±0.02
2.569±0.02
2.116±0.02
3.506±0.04
0.922±0.01
0.006±0.0001
0.104±0.002
0.437±0.006
0.411±0.005
0.325±0.004
0.297±0.001
0.433±0.003
0.114±0.002
0.602±0.004
0.746±0.004
0.508±0.004
0.414±0.005
0.922±0.01
0.874±0.004
0.823±0.01
0.650±0.004
0.594±0.01
0.867±0.004
0.228±0.002
1.203±0.01
1.492±0.02
1.016±0.01
0.829±0.01
1.818±0.02
1.261±0.01
0.07±0.0006
20
2.880±0.04
1.137±0.01
2.080±0.02
1.460±0.02
0.365±0.002
2.086±0.02
2.569±0.02
1.777±0.02
1.466±0.02
3.124±0.02
0.012±0.0001
0.052±0.0004
3.369±0.02
1.380±0.01
1.238±0.01
1.734±0.02
0.456±0.004
1.243±0.01
1.285±0.01
1.058±0.01
1.753±0.02
0.461±0.004
0.003±0.00002
0.052±0.0004
21
0.568±0.008
1.040±0.01
0.568±0.008
0.520±0.004
0.730±0.004
0.182±0.002
1.043±0.01
22
23
0.730±0.004
0.182±0.002
1.043±0.01
24
25
26
1.285±0.01
0.622±0.004
0.889±0.01
27
28
0.889±0.004
0.733±0.004
1.562±0.015
0.003±0.00002
0.026±0.0002
0.733±0.004
0.768±0.01
29
Ketoconazole
Itraconazole
0.003±0.00002
0.026±0.0002
0.433±0.005
0.035±0.0006
Experiments were performed in duplicate and repeated three times. Values are expressed as means ± SD (p < 0.05).
5
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Dataset collection and QSAR model analysis
position and influence of heteroatoms, play a critical role in
antifungal activity.[33] Particularly, the examination of these
descriptor's values showed that in the case of compound 29 the
sulfonamide group substitution is responsible for the significant
antifungal activity reduction. Furthermore, the predicted
conformation-independent aqueous solubility and the dipole
moment variables correlate positively and negatively to MIC
values, respectively. According to solubility values, compounds 5
(logS=-5.594) and 6 (logS=-5.388), bearing halogenated benzyl
rings, are more active compared to compound 1 (logS=-3.929).
The comparison of the dipole moment values of structurally
similar compounds 23 (dipole=5.027) and 24 (dipole=9.615) could
partially explain the higher activity of 24. To a lesser extent, the
number of nitrogen and oxygen atoms as well as the sum of their
topological distances and the Modified Randic connectivity
descriptor, also contribute to MIC values (Equation 1). Regarding
the observed differences in activity between tautomers 2 and 3,
and since compound 3 was excluded from the final model, these
could be attributed to electron affinity differences (0.55eV for
compound 2 and 0.095eV for compound 3). Electron affinity is a
parameter which has been correlated with anti-candidal activity of
N-heterocyclic thioamides.[34]
The antifungal activity values (Table 2) clearly indicate that the
examined compounds possessed higher antifungal activity
against Aspergillus fumigatus (strain ATCC9197) compared to
Candida albicans (strains 475/15, 10/15 and ATCC10231) and
Candida krusei (strain H1/16). Based on this observation the MIC
values against Aspergillus fumigatus were selected as dependent
variables to develop QSAR models. Specifically, MICs together
with molecular descriptors were employed as dependent and
independent variables, respectively and
a multiple linear
regression (MLR) analysis was performed to identify the structural
and/or physicochemical properties’ effects on the antifungal
activity. The QSAR analysis suggested a variety of models (see
Supporting Information) which were evaluated for reliability
according to statistically significant parameters including the
square of correlation coefficient (R2), the square of correlation
coefficient for cross validation (Q2), the sequential Fischer test (F-
test) and Root-Mean-Square Error (RMSE).
The most reliable model included all agents bearing 1H-1,2,4
triazole ring and a methylene linker in position 5, except
compounds 3 and 19 (Table 1). MLR analysis generated a model
described by a good correlation (R2=0.98) among 12 molecular
descriptors and the antifungal activity (Figure 1). The expression
of the key properties that may influence the antifungal activity
(Equation 1) and the statistically significant parameters obtained
from model analysis (Model 2), are described in Table 3. Also, the
crucial descriptor’s calculated values of the examined compounds
and the observed and predicted antifungal activity values
estimated by equation 1 are illustrated in Tables 4 and 5,
respectively.
Conclusions
We have developed a novel series of 1,2,4-triazole derivatives
that inhibit different strains of Candida albicans, Candida krusei
and Aspergillus fumigatus with low micromolar or submicromolar
MIC and MFC values. These inhibitors were derived from 1,2,4-
triazole ring by incorporating a variety of chemically diverse but
pharmacologically interesting groups in different positions of the
heterocycle. The tested compounds exhibited significant
antifungal activity, especially in the case of Aspergillus fumigatus,
where most of them proved to be equally or more active than
ketoconazole. The most active of them, compounds 24 and 5,
were 4 and 2-fold more active than ketoconazole. On the other
hand, these compounds were less active than itraconazole. Our
studies demonstrate that: (i) the fungicidal activity of this class of
compounds is greatly dependent upon the substitution on ring A;
(ii) amide substitution was mostly more favorable than urea or
halogen; (ii) addition of an extra aromatic ring (ring C) had a
positive influence on the potency against most of the fungi and
(iv) replacement of ring B with a heterocycle reduced potency.
For the case of Aspergillus fumigatus ATCC9197 strain, the
QSAR model revealed that the dipole-dipole nature of the
intermolecular forces, measured using the square of the dipole
moment divided by the molecular volume, (dipole2/V)
descriptor,[30] contributes significantly and positively to MIC values.
Thus, the higher dipole2/V values are the greater the MIC and
therefore the antifungal activity is decreased, such as in the case
of compound 18 (dipole2/V=0.047), which possessed weaker
antifungal activity compared to compounds 11 (dipole2/V=0.013)
and 16 (dipole2/V=0.007). The mean topological charge index of
order 9, which evaluates the charge transfers between pairs of
atoms and therefore the global charge transfers in the molecule[31]
also constitutes an important explanatory descriptor where an
increase in its value, reduces the MIC thus increases the
antifungal activity. Representatively, the descriptor’s value for
compound 6 which presents strong antifungal activity is 9.3 10-3,
compared to the corresponding value of the less active
compounds 2 and 1 (2.8 10-3). Another descriptor that emerges
from this model is the Path/Walk 3 - Randic shape index, which
describes the branching of the carbon-atom skeleton and also is
used to differentiate molecules based on their size, flexibility and
overall shape.[32] This descriptor contributes negatively to MIC
values, thus higher values demonstrate lower MIC hence
improved activity against Aspergillus fumigatus ATCC9197 strain,
such as in the case of compound 12 (0.333) compared to 25
(0.308). Also, the correlation analysis proved that the valence
connectivity indexes chi-3 and chi-4, which are related to the
Moreover, from the MLR analysis three interesting points arise.
Specifically, the MIC values significantly depend on: (i) the dipole-
dipole nature of the intermolecular forces (dipole2/V), (ii) the
global charge transfers (Mean topological charge index of order
9) and (iii) the branching of the carbon-atom skeleton (Path/Walk
3 - Randic shape index). Also, the descriptors of aqueous
solubility, dipole moment and the position and influence of
heteroatoms contribute, but to a lesser extent, on the activity.
Our findings suggest that the 1,2,4-triazole ring offers a promising
motif for further development of new analogues with optimized
antifungal properties through appropriate substitution.
6
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Table 3. Selected QSAR equation (Equation 1) and the statistical significance parameters, obtained from Model 2 analysis.
QSAR Model 2
N
R2
Q2
F-value
92.52
RMSE
0.10
Equation (1):
MIC 0.126(±0.011) logS
=
-
5.380(±1.181) Mean topological charge index of order
9
-
27
0.98
0.72
0.0074(±0.0006) Modified Randic connectivity + 0.039(±0.006) Number of N – 0.037(±0.007)
Number of - 0.0013(±0.0003) Sum of topological distance between N…O + 0.137(±0.009) Valence
connectivity index chi-3 + 0.575(±0.0382) Valence connectivity index chi-4 + 20.666(±4.17375)
Dipole2/V - 0.277(±0.042) Dipole - 2.346(±0.540) Path/walk 3 – Randic shape index + 1.22(±0.237)
Figure 1. Correlation plot of the observed versus predicted MIC (μmol·mL-1) values of triazole derivatives, including to Model 2. Compounds that comprise the
training and test set are illustrated with red and blue dots, respectively.
7
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Table 4. Predicted values of QSAR Model 2 significant descriptors.
Mean
topological
Charge
Index of order
9
Sum of
topological
distance
Modified
Randic
connectivity
Valence
connectivity
index chi-3
Valence
connectivity
index chi-4
Path/walk 3
– Randic
Shape index
Number
of N
Number
of O
Compounds
logS
Dip^ 2/V
Dipole
Between N..O
1
2
4
5
6
-3.929
-3.984
-4.313
-5.594
-5.388
2.8 10-3
2.8 10-3
7.9 10-3
6.9 10-3
9.3 10-3
55.015
54.811
61.518
65.546
65.989
3
3
3
3
3
0
0
1
0
0
0
0
2.796
2.784
3.100
3.383
3.664
1.887
1.879
2.027
2.149
2.253
0.033
0.021
0.022
0.033
0.046
5.311
4.177
4.441
5.44
0.317
0.317
0.327
0.320
0.332
22
0
0
6.518
7
8
-5.758
-5.01
6.7 10-3
69.319
67.056
3
3
0
1
0
3.652
3.423
2.327
2.164
0.006
0.006
2.444
2.318
0.338
0.328
10.6 10-3
22
9
-4.477
-6.607
-5.736
-7.12
10.2 10-3
6.1 10-3
6.6 10-3
6.2 10-3
8.0 10-3
5.7 10-3
4.8 10-3
8.2 10-3
63.002
78.148
81.958
93.136
87.496
84.857
96.655
87.496
4
3
4
4
4
4
4
4
0
0
1
1
1
1
1
1
0
3.243
4.325
4.189
4.876
4.475
4.386
5.357
4.511
2.056
2.867
2.782
3.355
3.012
2.976
3.728
2.920
0.008
0.006
0.013
0.061
0.026
0.037
0.012
0.007
2.79
0.321
0.329
0.327
0.333
0.327
0.317
0.327
0.327
10
11
12
13
14
15
16
0
2.632
3.899
8.768
5.689
6.803
4.041
2.993
30
30
30
30
30
30
-6.424
-6.015
-7.262
-6.424
17
18
20
-6.296
-5.468
-6.424
5.8 10-3
6.6 10-3
5.3 10-3
87.857
81.958
87.496
4
4
4
1
1
1
30
30
27
4.662
5.139
4.448
3.115
3.598
3.065
0.050
0.047
0.020
8.065
7.608
5.014
0.316
0.327
0.326
21
22
23
24
-5.468
-6.156
-6.85
5.7 10-3
6.9 10-3
6.7 10-3
7.0 10-3
85.265
90.804
96.342
96.342
5
5
5
5
1
1
1
1
32
32
32
32
4.185
4.507
4.756
4.793
2.804
2.942
3.269
3.177
0.052
0.028
0.019
0.071
7.964
5.942
5.027
9.615
0.317
0.319
0.318
0.319
-6.85
8
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25
-4.267
-4.045
-3.464
-5.121
-5.707
0.9 10-3
57.710
56.353
55.311
70.964
92.504
3
3
4
3
4
0
0
0
2
2
0
0
2.997
3.368
2.672
3.700
5.598
2.061
2.341
1.786
2.472
3.867
0.018
0.027
0.026
0.012
0.027
4.060
4.690
4.619
3.423
5.742
0.308
0.321
0.317
0.336
0.329
26
27
28
29
3.4 10-3
2.8 10-3
9.1 10-3
7.6 10-3
0
41
60
Table 5. Observed and Predicte dantifungal activity (MIC - μmol·mL-1) against Aspergillus fumigatus ATCC9197 by Model 2 QSAR equation.
Observed
Antifungal Activity
Predicted
Antifungal Activity
Compounds
ΔObs-Pred
Training Set
1
4
5
6
8
9
12
13
16
17
18
20
21
22
23
25
26
27
29
0.382
0.226
0.223
0.230
0.467
0.632
0.283
0.257
0.643
0.288
0.860
0.411
0.325
0.297
0.433
0.602
0.746
0.508
0.922
0.401
0.258
0.236
0.263
0.474
0.595
0.246
0.305
0.603
0.284
0.845
0.417
0.288
0.309
0.459
0.543
0.769
0.498
0.939
-0.019
-0.032
-0.013
-0.006
-0.007
0.037
0.037
-0.048
0.048
0.004
0.015
-0.006
0.037
-0.012
-0.026
0.059
0.023
0.010
-0.017
Test Set
2
7
0.340
0.497
0.636
0.282
0.298
0.765
0.114
0.414
0.454
0.523
0.720
0.512
0.307
0.833
0.198
0.446
-0.114
-0.026
-0.084
-0.230
-0.009
-0.068
-0.084
-0.032
10
11
14
15
24
28
9
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orange solid; 1H-NMR (300 MHz, CDCl3): δ (ppm): 4.13 (s, 2H, CH2), 7.28
(complex m, 5H, aromatic-H), 7.39 (complex m, 3H, aromatic-H), 7.96 (m,
2H, aromatic-H), 11.83 (br s, 1H, NH); elemental analysis: calcd for
C15H13N3: C 76.57, H 5.57, N 17.86; found: 76.39, H 5.47, N 17.96.
Experimental Section
Chemistry. General
3-(4-Methoxybenzyl)-5-phenyl-1H-1,2,4-triazole
(4):
Column
chromatography using CH2Cl2/MeOH 10/1 as eluents to give 219 mg
(83%) of a pure white solid; 1H-NMR (300 MHz, CDCl3): δ [ppm]= 3.69 (s,
3H, OCH3), 4.13 (s, 2H, CH2), 6.73 (d, 2H, J =11.6 Hz, aromatic-H), 7.08
(d, 2H, J =11.2 Hz, aromatic-H), 7.39 (complex m, 3H, aromatic-H), 7.94
(m, 2H, aromatic-H); elemental analysis: calcd for C16H15N3O: C 72.43, H
5.70, N 15.84; found: C 72.73, H 5.85, N 15.62.
Starting materials, reagents, and analytical grade solvents were
purchased from Sigma-Aldrich Europe (Germany). All reactions were
routinely checked by TLC using Merck Kieselgel 60 F254 aluminum plates
and visualized by UV light. The purity of all the intermediates was checked
by 1H NMR. ESI-MS analyses were performed on an Agilent 1100 LC–
MSD trap system VL. Flash chromatographic separations were performed
on a Biotage SP1 purification system using flash cartridges prepacked with
KP-Sil32–63 lm, 60 Å silica. Elemental analyses were performed on a
EuroEA 3000 analyzer; the measured values for C, H, and N had a
maximum deviation of ± 0.4% from the theoretical value. Melting points
(mp) were taken on a Gallenkamp MFB 595010M apparatus (open
capillary method). Nuclear magnetic resonance (NMR) spectra were
recorded at 300 MHz on a Varian Mercury 300 instrument at ambient
temperature in the specified deuterated solvent. Chemical shifts (d) are
quoted in parts per million (ppm) and are referenced to the residual solvent
peak. The coupling constants J are given in hertz (Hz). The following
abbreviations were used: s (singlet), d (doublet), t (triplet), q (quadruplet),
qn (quintuplet), dd (doublet of doublet), td (doublet of triplet), m (multiplet),
brs (broad signal). Signals due to NH/OH protons were located by
deuterium exchange with D2O.
3-(4-bromobenzyl)-5-phenyl-1H-1,2,4-triazole
(5):
Column
chromatography using CH2Cl2/MeOH 10/1 as eluents gave, after trituration
with PE/Et2O, 280 mg (89%) of a pure white solid; 1H-NMR (300 MHz,
CDCl3): δ [ppm]= 4.13 (s, 2H, CH2), 7.16 (d, 2H, J =11.2 Hz, aromatic-H),
7.43 (complex m, 5H, aromatic-H), 7.96 (m, 2H, aromatic-H, NH), 11.23
(br s, 1H, NH); elemental analysis: calcd for C15H12BrN3: C 57.34, H 3.85,
N 13.37; found: C 57.70, H 4.10, N 13.14.
3-(3,4-dichlorobenzyl)-5-phenyl-1H-1,2,4-triazole
(6):
Column
chromatography using CH2Cl2/MeOH 10/1 as eluents gave, after trituration
with MeOH, 260 mg (85%) of a pure crystalline white solid; 1H-NMR (300
MHz, CDCl3): δ [ppm]= 4.03 (s, 2H, CH2), 7.04 (d, 1H, J = 10.4 Hz,
aromatic-H), 7.36 (complex m, 5H, aromatic-H), 7.86 (d, 2H, J = 8.8 Hz
aromatic-H), 12.48 (br s, 1H, NH); elemental analysis: calcd for
C15H11Cl2N3: C 59.23, H 3.65, N 13.81; found: C 59.50, H 3.81, N 14.11.
1-Benzyl-4-phenyl-1H-1,2,3-triazole (1)[27]: Benzyl azide (125 μL, 1.0
mmol) and ethynylbenzene (110 μL, 1.0 mmol) were suspended in a 1:1
mixture of water and tert-butyl alcohol (4 mL). Sodium ascorbate (0.1 mmol,
100 μL of freshly prepared 1Μ solution in water) was added, followed by
copper (ΙΙ) sulfate pentahydrate (2.5 mg, 0.01 mmol, in 100 μL of water).
The heterogeneous mixture was stirred vigorously overnight, at which
point it cleared and TLC analysis indicated complete consumption of the
reactants. The reaction mixture was diluted with water (30 mL) and
extracted with ethyl acetate. The organic extracts were washed with water
and brine, dried (Na2SO4) and evaporate under vacuum. The residue was
purified by flash column chromatography, using as eluent CHCl3 to afford
200 mg (85%) of pure product as an off-white powder. 1H-NMR (300 MHz,
CDCl3): δ [ppm]= 5.58 (s, 2H, CH2), 7.32 (complex m, 3H, aromatic-H),
7.40 (complex m, 5H, aromatic-H), 7.66 (s, 1H, NH), 7.80 (m, 2H, aromatic-
H); elemental analysis: calcd for C15H13N3: C 76.57, H 5.57, N 17.86;
found: 76.22, H 5.40, N 18.03.
3-(2-chloro-6-fluorobenzyl)-5-(4-chlorophenyl)-1H-1,2,4-triazole (7):
Column chromatography using CHCl3, CHCl3/AcOEt, 7:3 as eluents gave
243 mg (65%) of a yellow solid;1H-NMR (300 MHz, CDCl3): δ [ppm]= 4.11
(s, 2H, CH2), 6.52 (s, H, NH), 7.08 (m, 1H, 3-aromatic-H), 7.30 (complex
m, 2H, 1,2-aromatic-H), 7.51 (d, 2H, J = 5.1 Hz 3,5-aromatic-H), 8.03 (d,
2H,
J = 5.1 Hz 2,6-aromatic-H); elemental analysis: calcd for
C15H10Cl2FN3: C 55.92, H 3.13, N 13.04; found: C 55.61, H 3.47, N 13.40.
5-(4-Chlorophenyl)-3-(4-methoxybenzyl)-1H-1,2,4-triazole (8): Column
chromatography using CHCl3, CHCl3/AcOEt, 7:3 as eluents gave 142mg
of a white pearl solid (55%); Rf 0.58 (AcOEt/ CH2Cl2, 25:75); mp: 152-
153 °C; 1HNMR (DMSO-d6): δ [ppm]= 3.69 (s,3H,CH3), 4.01 (s, 2H, CH2),
6.84 (d,2H, J = 8.8 Hz), 7.20 (d, 2H, J = 8.8 Hz), 7.47 (d, 2H, J = 8.4 Hz),
7.94 (d, 2H, J = 6.2 Hz), 13.91 (s,1H, NHtriazole); elemental analysis: calcd
for C16H14ClN3O: C 64.11, H 4.71, N 14.02; found: C 64.37, H 4.95, N
14.30.
5-Benzyl-3-phenyl-1H-1,2,4-triazole (3)[28]
:
2-Phenylacetohydrazide
(150.1 mg, 1.0 mmol) and benzonitrile (309 mg, 3.0 mmol) were
suspended in n-butanol (3 mL). Potassium carbonate (68 mg, 0.5 mmol)
was added and the heterogeneous mixture was stirred under microwave
irradiation at 150 °C for 3 h (TLC monitoring). The solvents evaporated
and the residue was purified with column chromatography using
CHCl3/MeOH 10/1 as eluents to give 140 mg (60%) of a pure white solid;
1H-NMR (300 MHz, CDCl3): δ [ppm]= 4.15 (s, 2H, CH2), 7.29 (complex m,
6H, aromatic-H, NH), 7.40 (complex m, 3H, aromatic-H), 7.98 (m, 2H,
aromatic-H); elemental analysis: calcd for C15H13N3: C 76.57, H 5.57, N
17.86; found: 76.34, H 5.38, N 18.00.
4-((5-(4-Chlorophenyl)-1H-1,2,4-triazol-3-yl)methyl)aniline (9): Column
chromatography using CHCl3, CHCl3/AcOEt, 7:3 as eluents gave 786mg
of a light yellow solid (50%); Rf 0.29 (AcOEt/ CH2Cl2, 9:1); mp: 189-191 °C;
1HNMR (DMSO-d6): δ [ppm]= 3.88 (s, 2H, CH2), 4.91 (s, 2H, NH2), 6.49
(d, 2H, J = 8.4 Hz), 6.93 (d, 2H, J = 8.0 Hz), 7.47 (d, 2H, J = 8.4 Hz), 7.93
(d, 2H, J = 6.5 Hz), 13.8 (s, NHtriazole); elemental analysis: calcd for
C15H13ClN4: C 63.27, H 4.60, N 19.68; found: C 63.50, H 4.34, N 19.95.
3-(Biphenyl-4-ylmethyl)-5-(4-chlorophenyl)-1H-1,2,4-triazole
(10):
Column chromatography using CHCl3, CHCl3/AcOEt, 7:1 as eluents gave
237mg of a white pearl solid (59%);Rf 0.29 (n-Hex 7:3); mp: 204-205 °C;
1HNMR (DMSO-d6): δ [ppm]= 4.10 (s, 2H, CH2), 7.32-7.49 (m,7H), 7.58-
7.63 (m,4H), 7.95 (d, 2H, J = 8.3 Hz), 14.02 (s, NHtriazole); elemental
analysis: calcd for C21H16ClN3: C 72.93, H 4.66, N 12.15; found: C 73.21,
H 4.86, N 12.32.
General procedure for the synthesis of triazole derivatives 2, 4, 5, 6,
7, 8, 9, 10, 30, 31 and 32.
The corresponding hydrazides (1.0 mmol) and benzonitriles (3.0 mmol)
were suspended in n-butanol (3 mL). Potassium carbonate (68 mg, 0.5
mmol) was added and the heterogeneous mixture was stirred under
microwave irradiation at 150 °C for 3 h (TLC monitoring) [700 W, with
temperature rising from ambient to 150 °C (5 min)]. The solvents
evaporated and the residue was purified with column chromatography.
3,5-Dibenzyl-1H-1,2,4-triazole (25): Column chromatography using
CHCl3, CHCl3/AcOEt, 7:1 gave 110mg of a white pearl solid (73%); Rf 0.57
(CH2Cl2/AcOEt, 6:4); mp: 146-148 °C; 1HNMR (300 MHz, DMSO-d6): δ
[ppm]= 3.90 (d, 4H, J = 23.4 Hz), 7.21-7.26 (m,10H), 13.46 (s, NHtriazole);
elemental analysis: calcd for C16H15N3: C 77.08, H 6.06, N 16.85; found: C
77.30, H 6.26, N 16.54.
3-Benzyl-5-phenyl-1H-1,2,4-triazole (2)[28]
using CH2Cl2/AcOEt 4/1 as eluents gave 200 mg (85%) of a pure light
: Column chromatography
10
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ChemMedChem
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3-Benzyl-5-(thiophen-2-yl)-1H-1,2,4-triazole
(26):
Column
2,4-dichloro-N-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)phenyl)
benzamide (12): Column chromatography using CH2Cl2, CH2Cl2/MeOH
10/1 as eluents gave 114mg of a white solid (69%); Rf 0.38 (CH2Cl2/AcOEt,
6:4); mp: 167-170 °C; 1HNMR (300 MHz, DMSO-d6): δ [ppm]= 4.06 (s, 2H,
CH2),7.27 (d, 2H, J = 8.4 Hz), 7.30-7.62 (m, 8H), 7.94 (d, 2H, J = 5.0 Hz),
10.68 (s, 1H, NHamid), 13.8 (s, 1H, NHtriazole); elemental analysis: calcd
for C22H16Cl2N4O: C 62.42, H 3.81, N 13.24; found: C 62.67, H 3.57, N
13.49.
chromatography using CHCl3, CHCl3/AcOEt, 7:3 gave 91 mg of a white
pearl solid (37%); Rf 0.69 (CH2Cl2/AcOEt, 6:4); mp: 138-139 °C; HNMR
(300 MHz, DMSO-d6): δ [ppm]= 4.06 (s, 2H, CH2), 7.11-7.30 (m, 6H), 7.54
(s, 2H, Hthiophene), 13.87 (s, NHtriazole); elemental analysis: calcd for
C13H11N3S: C 64.71, H 4.59, N 17.41; found: C 64.99, H 4.78, N 17.71.
1
4-(3-benzyl-1H-1,2,4-triazol-5-yl)pyridine
(27)[35]:
Column
chromatography using CHCl3, CHCl3/AcOEt, 7:3 gave 161 mg of a white
powder solid (64%); Rf 0.69 (AcOEt/MeOH, 9:1); mp: 197-201 °C; 1HNMR
(300 MHz, DMSO-d6): δ [ppm]= 4.12 (s, 2H, CH2),7.20-7.33 (m,1H), 7.25-
7.29 (m, 4H), 7.44-7.48 (m,1H), 8.25 (d, 1H, J = 8.4 Hz), 8.59 (d, 1H, J =
4.8 Hz), 9.12 (t, 1H, J = 1.4 Hz), 14.0 (s, NHtriazole); elemental analysis:
calcd for C14H12N4: C 71.17, H 5.12, N 23.71; found: C 71.17, H 5.12, N
23.71.
(N-(4-((5-(4-chlorophenyl)-1H-1,2,4-triazol-3-yl)methyl)phenyl)
benzamide (16): Column chromatography using CH2Cl2, CH2Cl2/MeOH
10/1 as eluents gave 39mg of a white solid (29%);Rf 0.37, (CH2Cl2/AcOEt,
6:4); mp 237-238 °C; 1HNMR (300 MHz, DMSO-d6): δ [ppm]= 4.05-4.1 (m,
2H, CH2), 7.25 (d, 2H, J = 8.3 Hz), 7.50-7.59 (m,5H), 7.69 (d, 2H, J = 8.3
Hz), 7.90 (d, 4H, J = 6.0 Hz), 10.2 (s, 1H, NHamid),13.90 (s, NHtriazol);
elemental analysis: calcd for C22H17ClN4: C 67.95, H 4.41, N 14.41; found:
C 68.25, H 4.67, N 14.70.
3-(Benzo[d][1,3]dioxol-5-ylmethyl)-5-(4-chlorophenyl)-1H-1,2,4-
triazole (28): Column chromatography using CHCl3, CHCl3/AcOEt, 7:3
gave 157mg of a white solid (55%); Rf 0.51 (AcOEt/CH2Cl2, 3:7); mp: 209-
210 °C; 1HNMR (300 MHz, DMSO-d6): δ [ppm]= 3.99 (s, 2H, CH2), 5.95
(s, 2H,CH2benzdioxole), 6.76 (d, 1H, J = 1.5 Hz), 6.86 (t, 2H, J = 7.5 Hz) 7.51
(dd, 2H, J = 2.2 Hz), 7.48 (d, 2H, J = 1.8 Hz), 13.02 (s, NHtriazole); elemental
analysis: calcd for C16H12ClN3O2: C 61.25, H 3.86, N 13.39; found: C 61.47,
H 3.70, N 13.50.
N-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)phenyl)cyclohexane
carboxamide (18): Column chromatography using CH2Cl2, CH2Cl2/MeOH
10/1 as eluents gave 15mg of a yellow solid (10%); mp: 222-225 °C;
1HNMR [300 MHz, (CD3)2CO]: δ [ppm]= 1.19-2.34 (m,10HcyHex), 4.25 (s,
2H, CH2), 7.29 (d, 2H, J = 8.8 Hz), 7.44-7.50 (m,3H), 7.62 (d,2H, J = 8.5
Hz), 8.05-8.32 (m, 2H), 9.00 (s,1H, NH); elemental analysis: calcd for
C22H24N4O: C 73.31, H 6.71, N 15.54; found: C 73.18, H 6.81, N 15.44.
4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)aniline
(30):
Column
chromatography using CHCl3, CHCl3/MeOH 9/1 as eluents gave 850 mg
(90%) of an off-white solid; 1H-NMR (300 MHz, CDCl3): δ [ppm]= 4.06 (s,
2H, CH2), 6.00 (br d, 2H, NH2), 6.62 (d, 2H, J = 11.2 Hz, aromatic-H), 7.05
(d, 2H, J = 11.2 Hz, aromatic-H), 7.41 (complex m, 4H, aromatic-H), 8.03
(m, 1H, aromatic-H), 11.23 (br s, 1H, NH); elemental analysis: calcd for
C15H14N4: C 71.98, H 5.64, N 22.38; found: C 72.30, H 5.95, N 22.71.
N-(3-((5-(3-chlorophenyl)-1H-1,2,4-triazol-3-yl)methyl)phenyl)
benzamide (19): When 32 reacted with benzoylchloride under the same
reaction conditions gave, after column chromatography of the residue
using ethyl acetate/n-hexane 2:1 v/v as the eluent,120mg of an oily
compound (61%); Rf 0.30 (CH2Cl2/Hex,6:1). The latter was dissolved in
HCl 1.25N in absolute ethanol to obtain its hydrochloride in pure form.
White powder; mp: 200-202 °C (dec.). 1H-NMR (300 MHz, DMSO-d6): δ
[ppm]=4.10 (s, 2H), 6.03 (brs, 2H, exch. D2O), 7.05 (d, 1H, J = 8.4 Hz),
7.29 (t, 1H, J = 8.4 Hz), 7.47-7.59 (m, 5H), 7.65-7.67 (m, 1H), 7.69 (s, 1H),
7.91 (d, 2H, J = 6.9 Hz), 7.97 (d, 2H, J = 8.4 Hz), 10.23 (s, 1H, exch. D2O).
IR (KBr film, cm-1) 3256, 3061, 2567, 1651, 1618, 1537, 1315, 698. ESI-
MS (M+H)+ 389.0, 391.0; elemental analysis: calcd for C22H18Cl2N4O: C
62.13, H 4.27, N 13.17; found: C 62.42, H 4.11, N 13.33.
3-((5-(3-Chlorophenyl)-1H-1,2,4-triazol-3-yl)methyl)aniline
(31):
Column chromatography using CHCl3, CHCl3/MeOH 9/1 as eluents gave
207mg of a yellow oil (62%); Rf 0.35 (AcOEt/ CH2Cl2,3:7); 1HNMR (CDCl3):
δ [ppm]= 3.69 (s, 2H,NH2), 4.06 (s,2H,CH2), 6.54-6.66 (m, 3H), 7.10 (t, 2H,
J = 7.7 Hz), 7.30-7.37 (m, 2H), 7.89-7.92 (m, 1H), 8.05 (s,1H, NHtriazole);
HRMS (m/z): [M+H]+ calcd for C15H13ClN4, 284.0829; found, 284.0838.
3-((5-(4-Chlorophenyl)-1H-1,2,4-triazol-3-yl)methyl)aniline
(32):
Column chromatography using CHCl3, CHCl3/MeOH 9/1 as eluents gave
207mg of a white powder (79%); C15H13ClN4; Rf 0.33 (AcOEt/CH2Cl2, 3:7);
1H-NMR (300 MHz, CDCl3): δ [ppm]= 4.09 (s, 2H, CH2), 6.70-6.72 (m, 2H),
7.18-7.20 (m, 2H), 7.33 (d, 2H, J = 8.5), 7.97 (d, 2H, J = 8.5 Hz); NH, NH2
not detectable; ESI-MS (M-H)- 282.8, 284.8 m/z; elemental analysis: calcd
for C15H13ClN4: C 63.27, H 4.60, N 19.68; found: C 63.07, H 4.98, N 19.89.
N-(3-((5-(3-chlorophenyl)-1H-1,2,4-triazol-3-yl)methyl)phenyl)
benzamide (20): When 31 reacted with benzoylchloride under the same
reaction conditions gave, after column chromatography of the residue
using CH2Cl2, CH2Cl2/MeOH 10/1, 120mg of a white foamy solid (63%); Rf
0.30 (CH2Cl2/Hex, 6:1); mp 59-61 °C; 1HNMR (300 MHz, CDCl3): δ [ppm]=
4.06 (s, 2H, CH2), 6.54-6.66 (m, 6H), 7.10 (t, 2H, J = 7.7 Hz), 7.30-7.37 (m,
3H), 7.89-7.92 (m,2H),10.2 (s, 1H, NHamid). ESI-MS (M+H)+ 389.0, 391.0;
HRMS (m/z): [M+H]+ calcd for C22H17ClN4O, 388.1091; found, 388.1086.
General procedure for the synthesis of triazole derivatives 11, 12, 16,
18, 19, 20 and 29.
4-Methyl-N-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)phenyl)
benzenesulfonamide (29): When 30 reacted with tosyl chloride under the
same reaction conditions gave, after column chromatography of the
residue using CH2Cl2, CH2Cl2/MeOH 10/1, 102mg of a white solid (66%);
Rf 0.32 (CH2Cl2/AcOEt, 6:4); mp: 208-212 °C; 1HNMR (300 MHz, DMSO-
d6): δ [ppm]= 2.29 (s, 3H, CH3), 3.96 (s, 2H, CH2), 6.98 (d, 2H, J = 8.7 Hz),
7.12 (d, 2H, J = 8.7 Hz), 7.28 (d, 2H, J = 7.6 Hz), 7.40-7.42 (m, 3H), 7.61
(d, 2H, J = 8.0 Hz), 7.90 (d, 2H, J = 6.5 Hz), 10.14 (s, 1H, NHsulphamid),
13.77 (s, NHtriazol); elemental analysis: calcd for C22H20N4O2S: C 65.33,
H 4.98, N 13.85; found: C 65.33, H 4.98, N 13.85.
The corresponding amine 30 / 31 / 32 / 9 (0.4 mmol) was dissolved in 5.5
mL of dry THF. The solution was cooled to 0 °C and three equivalents of
DIPEA (1.2 mmol) were added. Then the benzoylchloride (0.4 mmol) was
dissolved in 1.5 mL dry THF and added dropwise. The mixture was stirred
in rt for 5 h (TLC monitoring). The solvents evaporated and the crude
product was extracted with CH2Cl2 (3 x 20 mL), washed with water and
brine (2 x 20 mL) and dried over Na2SO4. The residue was purified with
column chromatography.
N-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)phenyl)benzamide (11)
General procedure for the synthesis of triazole derivatives 13, 14, 15
and 17
Column chromatography using CH2Cl2, CH2Cl2/MeOH 10/1 as eluents
gave 120 mg (86%) of a white solid;1H-NMR (300 MHz, CDCl3): δ [ppm]=
4.20 (s, 2H, CH2), 7.32 (m, 2H, aromatic-H), 7.53 (complex m, 7H,
aromatic-H), 7.87 (br d, 3H, J = 9.6 Hz, aromatic-H), 8.04 (br d, 2H, J =
10.8 Hz, aromatic-H); elemental analysis: calcd for C22H18N4O: C 74.56, H
5.12, N 15.81; found: C 74.27, H 5.40, N 16.01.
Equal amount of amine 30 (1eq) and the corresponding chlorobenzoic acid
(1eq), 2eq of N-methyl morpholine and 1.2eq of HOBt were dissolved in
dry THF and cooled to 0 °C in ice-bath and then 1.2eq of EDC was added.
The reaction was warmed to room temperature and stirred under argon
11
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10.1002/cmdc.202000312
ChemMedChem
FULL PAPER
flow overnight. The reaction mixture was extracted 3 times with brine and
CH2Cl2. Organic phases were combined and dried under sodium sulphate,
filtrated, and evaporated to dryness. The crude product was purified with
column chromatography.
calcd for C22H18ClN5O: C 65.43, H 4.49, N 17.34; found: C 65.80, H 4.21,
N 17.54.
N-(3-Chlorophenyl)-N1-[3-(5-(3-chlorophenyl)-1H-1,2,4-triazol-3-yl)
methyl]phenylurea (23): Column chromatography using ethyl acetate/n-
hexane 1:1 as eluents gave 48 mg of a white powder (53%); mp: 198-
200 °C; 1H-NMR (300 MHz, DMSO-d6): δ [ppm]= 4.08 (s, 2H), 6.92 (d, 1H,
J = 7.5 Hz), 6.98 (d, 1H, J = 7.5 Hz), 7.19-7.51 (m, 7H), 7.68 (s, 1H), 7.92-
7.94 (m, 1H), 7.96 (s, 1H), 8.86 (s, 1H, exch. D2O), 8.97 (s, 1H, exch. D2O);
NH not detectable. IR (KBr film, cm-1) 3292, 3063, 1642, 1591, 1554, 1226,
780. ESI-MS (M+H)+ 438.0, 440.0, 442.0; elemental analysis: calcd for
C22H17Cl2N5O: C 60.29, H 3.91, N 15.98; found: C 60.24, H 3.93, N 15.62.
3-Chloro-N-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)phenyl)
benzamide (13): Column chromatography using CH2Cl2/AcOEt, 7:3 gave
84mg of a white solid (60%); Rf 0.62 (CH2Cl2/AcOEt, 6:4); mp: 235-236 °C
1HNMR (300 MHz, DMSO-d6): δ [ppm]= 4.01 (s, 2H, CH2), 7.22-7.30 (m,
6H), 7.40-7.52 (m, 5H), 7.95 (d, 2H, J = 1.5 Hz), 10.11 (s, 1H,
NHamid),13.85 (s, NHtriazol); elemental analysis: calcd for C22H17ClN4O: C
67.95, H 4.41, N 14.41; found: C 67.66, H 4.09, N 14.58.
2-Phenyl-N-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)phenyl)
N-(3-Chlorophenyl)-N1-[3-(5-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl)
methyl]phenylurea (24): Column chromatography using ethyl acetate/n-
hexane 1:1 as eluents gave 50mg of a white powder, yield (53%); mp: 203-
205 °C. 1H-NMR (300 MHz, methanol-d4): δ [ppm]= 4.16 (s, 2H), 6.98-
7.03 (m, 2H), 7.20-7.39 (m, 7H), 7.46 (d, 2H, J = 9.0 Hz), 7.62 (s, 1H), 7.96
(s, 1H, exch. D2O), 7.99 (s, 1H, exch. D2O); NH not detectable. IR (KBr
film, cm-1) 3276, 1651, 1591, 1556, 1222. ESI-MS (M-H)- 435.8, 437.8,
439.9; elemental analysis: calcd for C22H17Cl2N5O: C 60.29, H 3.91, N
15.98;found: C 59.98, H 4.01, N 15.71.
acetamide (14): Column chromatography using CH2Cl2/AcOEt, 7:3 gave
64mg of a white solid (44%); Rf 0.32 (CH2Cl2/AcOEt, 6:4); mp: 233-
236 °C1HNMR (300 MHz, DMSO-d6): δ [ppm]= 4.06 (s, 4H, CH2), 7.29 (d,
3H, J = 8.4 Hz), 7.38-7.44 (m, 4H), 7.46-7.65 (m, 3H), 7.96 (dd, 4H, J =
4.1 Hz),10.30 (s, 1H, NHamid), 13.81 (s, NHtriazol); elemental analysis: calcd
for C23H20N4O: C 74.98, H 5.47, N 15.21; found: C 75.30, H 5.79, N 15.55.
2-(Naphthalen-2-yl)-N-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)
phenyl)acetamide (15): Column chromatography using CH2Cl2/AcOEt,
7:3 gave 120mg of yellow solid (72%); Rf 0.23 (CH2Cl2/AcOEt, 6:4); mp:
233-236°C; 1HNMR (300 MHz, DMSO-d6): δ [ppm]= 3.78 (s, 2H, CH2),
4.02 (s, 2H, CH2), 7.23-7.30 (s, 2H, J = 8.2 Hz), 7.37-7.48 (m, 8H), 7.83-
7.95 (m, 6H), 10.19 (s,1H,NHamid),13.85 (s, NHtriazol); elemental analysis:
calcd for C27H22N4O: C 77.49, H 5.30, N 13.39; found: C 77.77, H 5.11, N
13.08.
Antifungal activity evaluation
Strains C. albicans 475/15, C. albicans 10/15 and C. krusei H1/16 were
isolated from oral cavities of patients at Ear, nose and throat clinic, Clinical
hospital center, Zvezdana Serbia. They were identified on CHROMagar
plates and maintained on Sabouraud Dextrose Agar. Reference strains C.
albicans ATCC 10231 and A. fumigates ATCC 9197 were also used in this
study. Minimal inhibitory (MIC) and minimal fungicidal (MFC)
concentrations were determined according to Smiljkovic et al.[36] The MIC
values were considered as the lowest concentrations without
microscopically observed fungal growth after 24 h incubation at 37ꢀ°C for
Candida strains and 72 h at 28 °C for A. fumigatus. Following the serial
subcultivations of 10 μL into microtiter plates containing 100 μL of broth
per well, as well as subsequent incubation at 37ꢀ°C for 24 h (Candida sp.)
and 28°C for 72 h (A. fumigatus), the lowest concentrations with no visible
growth were defined as the MFC values, indicating 99.5ꢀ% killing of the
original inoculum.
3-phenyl-N-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)phenyl)
propanamide (17): Column chromatography using CH2Cl2/AcOEt, 7:3
gave 145mg of a white solid (63%); Rf 0.54 (CH2Cl2/AcOEt, 7:3); mp: 198-
200 °C1HNMR (300 MHz, DMSO-d6): δ [ppm]= 2.57 (t, 2H, PhCH2CH2-CO,
J = 8.0 Hz), 2.87 (t, 2H, PhCH2CH2-CO, J = 8.1 Hz), 3.94 (s, 1H, CHA),
3.99 (s, 1H, CHB), 7.12-7.27 (m, 7H), 7.38-7.51 (m, 5H), 7.94 (d, 2H, J =
6.9 Hz) ,9.86 (s,1H, NHamid),13.78 (s, NHtriazol); elemental analysis: calcd
for C24H22N4O: C 75.37, H 5.80, N 14.65; found: C 75.72, H 5.49, N 14.93.
General procedure for the synthesis of triazole derivatives 21, 22, 23
and 24
Molecular descriptors calculations and Quantitative structure-
activity relation (QSAR) modeling
The corresponding amine 30 / 32 (0.4 mmol) was dissolved in 3 mL of dry
THF. The solution was cooled to 0 °C and triethylamine (0.4 mmol) was
added. Then the corresponding phenylisocyanate (0.4 mmol) was
dissolved in 3 mL dry THF and added dropwise. The mixture was stirred
in rt for 48 h (TLC monitoring). The mixture was quenched with water and
extracted with AcOEt (3x20 mL), washed with water and brine (2x20 mL)
and dried over Na2SO4. The residue was purified with column
chromatography.
The 29 tested triazole derivatives (Table 1) were sketched in 2D form and
then were converted in 3D form and prepared in pH 7.0 ± 0.5 using
LigPrep[37] program of MAESTRO software.[38] The cheminformatics
program Canvas[39] was carried out to build a pool of molecular descriptors,
including physicochemical, constitutional and topological descriptors.
Totally, 100 descriptors were calculated and were utilized as the
dependent parameters of the QSAR modeling. The observed MIC and
MFC values (Table 2) indicated that the examined derivatives possessed
higher antifungal activity against Aspergillus fumigatus ATCC9197 strain,
in comparison to the other 4 strains. Therefore, the MIC values of this
strain were selected as the dependent variable for the QSAR models
development. In the present study, the Multiple Linear Regression (MLR)
analysis was implemented, using Canvas program,[36] to quantify the
relationship between linear combinations of the dependent variable MIC
and the statistical significance molecular descriptors. The validation of the
derived QSAR models is necessary in order to identify and evaluate their
predictive ability. The synthesized compounds, comprising the dataset of
the present study, are divided randomly into training set (70%) responsible
for model construction and test set (30%) responsible for model validation.
For the assessment of statistical quality of all QSAR derived models
several validation parameters were employed. Especially, in QSAR model
summary, N is the number of compounds, R2 is the squared correlation
coefficient (goodness of fit), Q2 is the predictability coefficient, F-value is
1-Phenyl-3-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)phenyl)urea
(21): Column chromatography using CH2Cl2, CH2Cl2/MeOH 10/1 as
eluents gave 120 mg (86%) of a white solid; 1H-NMR (300 MHz, DMSO-
d6): δ [ppm]=4.11 (s, 2H, CH2), 6.98-7.03 (m, 1H), 7.19-7.29 (m, 5H), 7.36-
7.44 (m, 5H), 7.68 (s,1H), 7.94 (dd, 2H, J = 6.3 Hz), 8.82 (s, 1H, exch. D2O,
NHamid), 8.93 (s, 1H, exch. D2O, NHamid), 13.81 (s, 1H, NHtriazole); elemental
analysis: calcd for C22H19N5O: C 71.53, H 5.18, N 18.96; found: C 71.15,
H 5.00, N 19.30.
1-(4-Chlorophenyl)-3-(4-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)
phenyl)urea (22): Column chromatography using CH2Cl2, CH2Cl2/MeOH
10/1 as eluents gave 100mg of a yellow solid (80%)’; Rf 0.46 (MeOH/
CH2Cl2,1:9); mp: 205-209 °C; 1H-NMR (300 MHz, DMSO-d6): δ [ppm]=
4.01 (s, 2H, CH2), 6.98-7.03 (m, 1H), 7.19-7.29 (m, 5H), 7.36-7.44 (m, 4H),
7.68 (s,1H), 7.94 (dd, 2H, J = 6.3 Hz), 8.82 (s, 1H, exch. D2O, NHamid), 8.93
(s, 1H, exch. D2O, NHamid), 13.81 (s, 1H, NHtriazole); elemental analysis:
12
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10.1002/cmdc.202000312
ChemMedChem
FULL PAPER
the Fischer ration between the variances of calculated and observed
activities and RMSE is the root mean square error.
[29] S. S. Goher, K. Griffett, L. Hegazy, M. Elagawany, M. M. H. Arief, A. Avdagic,
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Acknowledgements
[32] I. Gutman, O. Milijkovic, G. Caporossi, P. Hansen, Chem. Phys. Lett. 1999,
306, 366-372.
G. Z. would like to thank the Empirikion Foundation for the
financial support. M. S. and M. I. are grateful to the Serbian
Ministry of Education, Science and Technological Development
for financial support (451-03-68/2020-14/200007).
[33] X. H. Li, M. Solimannejad, J. Mol. Struc. Theochem. 2003, 663, 81-85.
[34] J. Stachowicz, E. Krajewska-Kułak, C. Lukaszuk, A. Niewiadomy, Indian J.
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[35] E. J. Browne, J. B. Polya, J. Chem. Soc. C 1968, 2904-2908
[36] M. Smiljkovic, M. T. Matsoukas, E. Kritsi, U. Zelenko, S. G. Grdadolnik, R.
C. Calhelha, I. C. F. R. Ferreira, S. Sankovic-Babic, J. Glamoclija, T.
Fotopoulou, M. Koufaki, P. Zoumpoulakis, M. Sokovic, ChemMedChem
2018, 13, 251-258.
Keywords: Azoles • Triazoles • Synthesis • Antifungal Activity •
Aspergillus • Candida• MIC values • SAR • QSAR
[37] LigPrep, Schrödinger, LLC, New York, NY, 2018.
[38] Maestro, Schrödinger, LLC, NY, 2018.
References:
[39] Canvas, Schrödinger, LLC, New York, NY, 2018.
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10.1002/cmdc.202000312
ChemMedChem
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Triazole, the magic ring: The present work has extended the structure–activity relationships of substituted 1,2,4-triazole derivatives
in determining in vitro inhibitory and fungicidal activity. Our studies demonstrate that the antifungal activity of this class of compounds
is greatly dependent upon the substitution on either the aromatic ring A or C. Multiple linear regression (MLR) analysis revealed three
critical descriptors that contribute on the activity.
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This article is protected by copyright. All rights reserved.