Design, synthesis and evaluation of hydrazine and acyl hydrazone derivatives of 5-pyrrolidin-2-one as antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2020.127220

Twenty-eight 5-pyrrolidine-2-ones decorated by hydrazine or acyl hydrazones groups have been designed, synthesized and evaluated as antifungal agents on a panel of twelve fungal strains and three non albicans candida yeasts species which have demonstrated reduced susceptibility to commonly used antifungal drugs. Half of the target compounds exhibited good to high antifungal activities on at least one strain with MIC₅₀ lower than the control antifungal agent – hymexazol or ketoconazole. 5-Arylhydrazino-pyrrolidin-2-ones were found active and the –NH-NH- linker proved to be essential to maintain the antifungal potential. Compound 2a is a broad-spectrum antifungal, active on 60% of the tested strains. Replacing the hydrazine linker by an acylhydrazone one narrowed the spectrum of activity but pyroglutamylaryl hydrazones, mainly aromatic ones, exhibited good activity, adequate “fungicide-like” properties and were devoted of cytotoxicity.

Full text of DOI:10.1016/j.bmcl.2020.127220

Bioorganic&MedicinalChemistryLetters30(2020)127220
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of hydrazine and acyl hydrazone
derivatives of 5-pyrrolidin-2-one as antifungal agents
Anca-Elena Dascalua,b,c,d, Alina Ghineta,b,d, Emmanuelle Lipkaa,c, Christophe Furmana,e
,
⁎
Benoît Rigoa,b, Antoine Fayeullef, Muriel Billamboza,b,
a Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1167 – RID-AGE – Facteurs de risque et déterminants moléculaires des maladies liées au vieillissement, F-59000
Lille, France
b Laboratoire de Chimie Durable et Santé, Health & Environment Department, Team Sustainable Chemistry, Ecole des Hautes Etudes d’Ingénieur (HEI), Yncréa Hauts-de-
France, 13 Rue de Toul, F-59046 Lille, France
c UFR Pharmacie, Laboratoire de Chimie Analytique, BP 83, F-59006 Lille, France
d ‘Alexandru Ioan Cuza’ University of Iasi, Faculty of Chemistry, Bd. Carol I nr. 11, 700506 Iasi, Romania
e Institut de Chimie Pharmaceutique Albert Lespagnol, 3 Rue du Professeur Laguesse, F-59000 Lille, France
f Sorbonne University, Université de Technologie de Compiègne, ESCOM, EA 4297 TIMR, Centre de recherche Royallieu, CS 60319, 60203 Compiègne cedex, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
Twenty-eight 5-pyrrolidine-2-ones decorated by hydrazine or acyl hydrazones groups have been designed,
synthesized and evaluated as antifungal agents on a panel of twelve fungal strains and three non albicans candida
yeasts species which have demonstrated reduced susceptibility to commonly used antifungal drugs. Half of the
target compounds exhibited good to high antifungal activities on at least one strain with MIC50 lower than the
control antifungal agent – hymexazol or ketoconazole. 5-Arylhydrazino-pyrrolidin-2-ones were found active and
the –NH-NH- linker proved to be essential to maintain the antifungal potential. Compound 2a is a broad-
spectrum antifungal, active on 60% of the tested strains. Replacing the hydrazine linker by an acylhydrazone one
narrowed the spectrum of activity but pyroglutamylaryl hydrazones, mainly aromatic ones, exhibited good
activity, adequate “fungicide-like” properties and were devoted of cytotoxicity.
Antifungal agents
Biological screening
Pyrrolidin-2-one
Hydrazone
Hydrazine
In the past decade, some molds and phytopathogenic fungal species
e.g. from the Aspergillus, Fusarium or Alternaria genera, have proved to
cause diseases in man, such as allergies, respiratory problems or in-
fections. The effects of toxins produced by these pathogens have am-
plified and emerged as a common threat to both agricultural produc-
tion, animal and human healths.1,2,3,4 Indeed, this contamination by
fungi and molds has an economic impact due to crops infestation,
quality decrease of agricultural products and impact on health of hu-
mans and animals feed with contaminated food.5,6 As a consequence,
innovative antifungal agents targeting agricultural fungal strains and
yeasts are needed. L-Pyroglutamic acid (PGA) 1 naturally occurred from
the cyclisation of glutamine and glutamic acid issued from the beet root
(Beta vulgaris saccharifera).7,8 The structural features of 1 and other
natural lactams9–11 are wide and exhibit a large range of biological
activities (Fig. 1).12–14 On the other hand, hydrazones are frequently
found in natural sources such as plants,15 fungi16 and marine organ-
isms.17 Natural and synthetic hydrazones are part of compounds dis-
antimicrobial ones18–20 while naturally occurring hydrazine derivatives
are very scarce but also of biological importance.21 Considering the
importance of these building blocks, hydrazine or acylhydrazone
functionalities were linked to a γ-lactam ring.
Thus, two series of compounds (Series 2 and 3, Fig. 1 and Scheme
1), issued from L-pyroglutamic acid were designed and evaluated for
their antifungal properties on a large panel of fungi and molds.
Target compounds 2a-e have been obtained by a two-steps route via
pterolactam (5-methoxypyrrolidinone) 4 (Scheme 1). To the best of our
knowledge, the general scaffold of hydrazines 2 was not reported in
literature. Starting from
decarboxylative oxidation Lf-uprynriosghleudtarmacicemaiccidpte1r,olaanctaemlec4troinchqeumainctai-l
tative yield.22 We recently described a new procedure for the reaction
of 4 with nucleophiles in non-acidic conditions.23 By using this method,
aromatic hydrazines and a catalytic amount of cesium fluoride were
reacted under a moderate vacuum (30 mmHg), with lactam 4 at 80 °C
for 6 to 19 h. In these conditions, racemic N,N’-aminals 2a,b and 2d,e
were obtained in good 60–85% yields; however, because of purification
playing
a
large spectrum of biological properties including
⁎ Corresponding author.
E-mail address: muriel.billamboz@yncrea.fr (M. Billamboz).
https://doi.org/10.1016/j.bmcl.2020.127220
Received 24 February 2020; Received in revised form 7 April 2020; Accepted 25 April 2020
Availableonline28April2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

A.-E. Dascalu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127220
Table 1
Antifungal activity of pyrrolidin-2-one derivatives at 100 μg/mL.
Compound Average inhibition rate SD (%) (n = 3)
FS PV PO AO BC AA CC SS GC
100 78 90
CK CP CT
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
3f
71
0
62
0
82
72
2
44
29
81
42
0
76
0
35
20
51
20
0
96 89
65 62
38 51
61 20
95
17 73
28 25
40 22
46 18
100 82 35
100 70 56
37
45
23
17
0
39
20
0
0
64
43
72
0
11 21
0
0
32
5
0
8
8
0
0
27
0
0
27
0
15
0
0
38
45 28
42
0
88
0
66
0
28 38
39
0
4
0
0
0
18
0
0
0
31 45
46 25
14 30
0
13
0
0
56
12
0
0
39
83
18
40
38
37
0
0
59
40
0
0
20
38
20
8
0
7
15
7
7
0
21 70
18
0
3g
3h
3i
22
0
0
0
0
0
0
0
0
0
0
36 23
39 29
27 92
13 22
19 44
37 26
8
8
1
62
68
30
0
0
0
6
0
53
98
12
36
34
11
11
72
40
0
0
12 17
50 47
32 16
3j
61
60
0
0
34
0
20
0
58 92
58 73
26
0
3k
3L
3m
3n
3o
3p
3q
3r
12
4
0
12
0
0
0
5
0
62
62
36
85
47
47
0
18
18
0
0
0
0
58
40
55
100
13
4
6
Fig. 1. Natural compounds containing lactam or hydrazine/hydrazone moi-
0
21
17
0
0
eties.
44 44
77
0
21
0
21
0
21 17
0
43
0
40
0
37 100
2
92
100 20 14
30
0
9
30
22
0
38 42
35 19
difficulties, the para-trifluoromethyl analogue 2c was isolated in a
lower 20% amount. These lactams, in which the pyrrolidin-2-one ring is
directly attached to an aryl hydrazine will be referred as type I deri-
vatives. The general structure of hydrazones 3 can be found in litera-
ture, as hypertensive agents24 or because of their central nervous
system stimulating activity.25 Target compounds 3a-w were obtained in
middle to very good yields (23–97%) by synthesizing firstly methyl
pyroglutamate 626 which was converted to the known pyroglutamyl
hydrazide 527 before reaction with the corresponding aldehydes in
water at room temperature.28 This series of compounds, in which a
carbonyl group is inserted between the lactam ring and a hydrazone
moiety, will be named type II. Derivatives type IIA (3a,c,d,h,i) dis-
tinguished from IIB (3b, 3e-g, 3j-w) by the presence of an aliphatic
chain; for IIB, the hydrazone moiety is attached to an aromatic or
heterocycle ring.
100
0
3
13
32
0
27
0
21 50
3s
33 20
42
0
0
0
0
0
10
28
40
50
2
0
0
3t
94
0
35
0
11
24
0
0
5
28
32 33
40 49
3u
3v
3w
36 23
26 32
42 62
0
3
35
0
38 25
0
0
29
0
0
0
0
18
10
0
0
23
0
0
33
72
FS – Fusarium solani, PV - Paecilomyces variotii, PO – Penicillium ochro-
chloron, AO – Aspergillus oryzae, BC – Botrytis cinerea, AA – Alternaria al-
ternata, CC – Cladosporium cladosporioides, SS – Sclerotinia sclerotiorum, GC –
Geotrichum candidum, CK – Candida krusei, CP – Candida pseudotropicalis, CT
– Candida tropicalis.
evaluated using the micro-dilution method, by using hymexazol, flu-
conazole and ketoconazole as the positive controls. A panel of twelve
agricultural fungi (Fusarium solani, Paecilomyces variotii, Penicillium
ochrochloron, Aspergillus oryzae, Alternaria alternata, Cladosporium
The in vitro antifungal activities of these original compounds were
Scheme 1. Reagents and conditions: (a) electrochemical decarboxylation, quant. yield;22 (b) 1 eq of H2N-N(R1,R2), CsF (5 mol%), solvent-less, 6–20 h, 80 °C;23 c)
CH3SO3H, MeOH/ CHCl3, MS 3 Å, reflux, quant. yield;26 d) hydrazine monohydrate;27 (e) aldehyde (1 equiv.), H2O, rt, 1 h.
2

A.-E. Dascalu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127220
Table 2
MIC50 values of the active compounds 2 and 3 against 10 fungal strains.a
Compounds
MIC50 (µg/mL)
Spectrumb
FS
PV
PO
AO
AA
CC
SS
GC
CP
CT
2a
18.5
24.5
11.7
31.0
> 75
/
7.8
4.4
43.4
3.6
/
23.0
/
0.6
/
58.5
8
3
3
2
0
0
1
3
1
1
4
1
1
1
1
–
2b
/
21.1
/
18.6
/
/
/
/
2c
2.0
/
/
/
3.4
/
/
/
/
2d
14.8
/
/
/
/
/
/
/
/
3b
/
/
/
/
/
/
> 75
> 75
62.0
1.8
1.8
63.7
5.9
/
/
/
3e
/
/
/
/
/
/
/
/
3f
/
/
/
/
/
/
/
/
/
3j
/
/
15.4
/
/
23.9
/
/
/
3k
/
/
/
/
/
/
/
/
/
3o
/
/
/
/
/
/
/
/
/
3p
6.5
59.1
51.6
101.5
/
/
5.3
/
/
53.0
/
/
> 75
3q
/
/
/
/
/
/
/
/
3r
/
/
/
/
/
/
/
/
/
3t
/
/
/
/
/
/
/
/
/
3w
/
/
/
/
/
/
38.4
1.5
> 50
1.6
/
/
Ketoconazolec
Hymexazol
Fluconazoled
/
/
/
/
/
/
/
9.6
/
15.9
15.6
/
57.2
/
62.2
/
45.7
/
38.3
/
28.9
/
26.5
/
/
/
–
/
–
FS – Fusarium solani, PV - Paecilomyces variotii, PO – Penicillium ochrochloron, AO – Aspergillus oryzae, AA – Alternaria alternata, CC – Cladosporium clados-
porioides, GC – Geotrichum candidum, CP – Candida pseudotropicalis, CT – Candida tropicalis.
a
b
c
d
Table 2 only summarized the compounds which exhibited antifungal activities against at least one strain among the tested panel of strains.
Spectrum refers to the number of strains killed by the compound.
Ketoconazole was used as positive control for G. candidum, C. pseudotropicalis and C. tropicalis bioassay.
Fluconazole was used as positive control for G. candidum and C. krusei bioassay.
cladosporioides, Geotrichum candidum, Sclerotinia sclerotiorum and
Botrytis cinerea) known for causing allergies, asthma and mycosis, and
also non albicans Candida yeasts species (Candida pseudotropicalis,
Candida tropicalis and Candida krusei) which have demonstrated re-
duced susceptibility to commonly used antifungal drugs was selected.29
The results of the first screening at a concentration of 100 µg/mL were
listed in Table 1. To further evaluate the inhibitory potencies of the
most promising synthesized compounds, the half maximal inhibitory
concentration (MIC50) values of products with high inhibition rate
(> 70%) were determined.
but did not inhibit the growth of the other species. Moreover, 2c is the
only one towards which C. cladosporioides is sensitive, with MIC50 of
3.4 µg/mL, eight-time more active than the positive control hymexazol
(Table 2, compound 2c).
Nine on sixteen acylhydrazones from type IIB, obtained from aro-
matic aldehydes, presented interesting activities, but with a narrower
spectrum of antifungal potency. All compounds showed no activity
against P. variotii, A. oryzae, A. alternata, C. pseudotropicalis or C. tro-
picalis. Interestingly, para substitution of the phenyl ring by a chloride
or bromine atom led to compounds 3j and 3p respectively, with a
broader spectrum, and activities better than hymexazol against F. solani
and C. cladosporioides. The electrodonating OCH3 substituent in the para
position (3u, 3v) abolished the global antifungal activity (Table 2, 3u,
3v). Exchanging the para-Br of 3p for a CF3, CH3 or NO2 group (3o, 3t,
3q) reduced its activity against F. solani. Interestingly, moving the para-
NO2 group of 3q to the ortho position led to 3r which maintained its
activity against the same fungi. In this series, poly-substitution gen-
erally dramatically decreased the activities, except for 3 k which pre-
sented the same good activity as 3j (~1.8 µg/mL) against G. candidum,
being three times more efficient than the para-bromo compound 3p. G.
candidum proved to be rather sensitive to the aryl hydrazones of the
series IIB because six on nine of these products inhibited its growth,
three of this series being as potent as the controls (Table 2, compounds
3j, 3k, 3p). Even the ortho-OH and para-CF3 phenyl (3f, 3o) as well as
the chloro-pyridyl (3w) compounds which were inactive against all the
other strains, led to a middle inhibition of its growth at 38–64 µg/mL
(positive control ketoconazole: 1.6 µg/mL). It is also notable that for
compounds 3b and 3e, the sequential dilution for inhibition of G.
candidum growth led to a dramatic decrease in activity (< 10% in-
hibition at 50 µg/mL).
In term of antifungal activity (Table 2), half of the newly synthe-
sized compounds displayed antifungal activities against at least one
strain. First, it is notable that all aliphatic hydrazone derivatives (type
IIA) displayed poor antifungal activity (Table 2, entries 3a, 3c, 3d, 3h),
and all active compounds exhibit an aromatic or heterocyclic ring.
Noteworthy also, all products in this series displayed no activity against
B. cinerea, S. sclerotiorum and C. krusei. Concerning hydrazine deriva-
tives type I, all compounds but 2e, bearing a 2-pyridyl moiety, dis-
played good antifungal properties, especially against F. solani and P.
variotii (Table 2, 2a–d). 5-(2-Phenylhydrazinyl)pyrrolidin-2-one 2a
showed a broad antifungal activity being able to fight against 8 strains
e.g. F. solani, P. variotii, P. ochrochloron, A. oryzae, A. alternata, S.
sclerotiorum, C. pseudotropicalis and C. tropicalis, with MIC50 values
ranging from 0.6 to 58.5 µg/mL.
Changing R1 from a hydrogen atom to a methyl group narrowed the
spectrum of activity underlining the importance of the -NH-NH- linker
to have a broad spectrum antifungal (Table 2, compound 2d). Inter-
estingly, replacing the phenyl group of 2a by a 2-pyridinyl heterocycle
led to a complete loss of the antifungal activity (Table 2, compound 2e).
Furthermore, substitution of the phenyl ring of 2a led to more selective
compounds targeting only two or three fungal strains. Thus, addition of
two fluorine atoms in the 2- and 5-position in 2b conserved the activity
against F. solani, but this compound was 4-fold less active against P.
ochrochloron and A. alternata, but still equivalent to that of the positive
control Hymexazol, while the activity against A. oryzae, C. pseudo-
tropicalis and C. tropicalis was lost. Compound 2c, substituted by a p-
To further evaluate their potential for candidate lead fungicide,
some of the molecular physicochemical properties [octanol/water -
partition coefficient (LogP), topology polar surface area (TPSA),
number of hydrogen bond donor sites (NDS), number of hydrogen bond
acceptors sites (NAS) and molecular weight (MW)] were calculated
(Table 3),30 with MarvinSketch 18.24, a ChemAxon software. All these
compounds met the Lipinski “Rule of five” criteria31 with Log P < 1.8,
NDS < 4, NAS < 9 and MW < 310, and the Briggs “Ground rules of
trifluoromethyl group displayed
a
narrow impressive activity
(2.0–11.7 µg/mL) specific for F. solani, P. variotii and C. cladosporioides
3

A.-E. Dascalu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127220
Table 3
scholarship. The authors gratefully acknowledge the National Cancer
Institute (NCI) for the biological evaluation of compounds on their 60-
cell panel: the testing was performed by the Developmental
Therapeutics Program, Division of Cancer Treatment and Diagnosis (the
URL to the Program’s website: http://dtp.cancer.gov).
Predicted molecular properties of the new compounds.33
Compound
Log Pa
TPSAb
NDSc
NASd
MWe
Spectrum
2a
2b
2c
2d
3f
0.93
1.22
1.81
0.98
0.14
1.05
1.65
1.32
1.21
0.38
0.38
0.96
0.17
53.16
53.16
53.16
44.37
90.79
70.56
70.56
70.56
70.56
116.38
116.38
70.56
83.45
3
3
3
2
3
2
2
2
2
2
2
2
2
4
4
4
4
7
5
5
5
5
9
9
5
6
191.23
227.21
259.23
205.26
247.25
265.70
300.14
299.25
310.15
276.25
276.25
245.28
266.68
7
3
3
2
1
3
1
1
4
1
1
1
1
Appendix A. Supplementary data
3j
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127220.
3k
3o
3p
3q
3r
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side products were observed for this type of reaction.
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analyzed: 1) 5-arylhydrazino pyrrolidin-2-ones 2 were found active; 2)
the –NH-NH- linker proved to be essential to maintain the antifungal
potential; 3) pyroglutamylaryl hydrazones 3, mainly aromatic ones,
exhibited good activity, adequate “fungicide-like” properties and were
devoted of cytotoxicity. In particular, these series of compounds proved
to be novel fungicide candidates to fight F. solani and G. candidum, two
strains which are known to pose health risks related to major barrier
breaks among people with weak immune systems.
29. Yiqing T, Jianguo T. Microbio Res. 2017;198:27–35.
30. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Adv Drug Deliv Rev. 1997;23:3–25.
31. Briggs GG. ‘Predicting uptake and movement of agrochemicals from physical prop-
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33. The herein calculations were performed with MarvinSketch 18.24, a ChemAxon
software.
34. The human embryonic kidney 293 cell line (HEK293) was cultured in Dulbecco’s
modified Eagle medium (DMEM) (Gibco, Waltham, MA) supplemented with 2 mM L-
glutamine, 100 IU/ml penicillin/streptomycin, non-essential amino acid solution (1/
100) and 5% (v/v) heat-inactivated foetal bovine serum (Sigma-Aldrich, Saint-Louis,
MO), and grown at 37 °C in a humidified incubator with 5% CO2. Cells were seeded
at 3000 cells per well onto 96-well plates in DMEM medium. Cells were incubated in
a culture medium that contained 100 µM of the different test compounds and 2 µM of
the references, each dissolved in less than 1% DMSO. After 72 h of incubation, cell
viability (in proliferation and cytotoxicity) was estimated by the colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium) assay.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
A.E.D. is grateful to the Fondation de la Catho for her PhD-
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