Design, synthesis and antifungal activity of pterolactam-inspired amide Mannich bases

Article abstract of DOI:10.1016/j.fitote.2020.104581

Pterolactam (5-methoxypyrrolidin-2-one) is a heterocycle naturally occurring in plants. In an attempt to identify antifungal agents, a series of novel Mannich bases of amide derivated from Pterolactam have been designed, synthesized and their antifungal activities were evaluated on a panel of nine fungal strains and three non albicans candida yeasts species which have demonstrated reduced susceptibility to commonly used antifungal drugs. A third of the target compounds exhibited good to high antifungal activities on at least one strain with EC₅₀ lower than the control antifungal agent. N,N′-aminals derivated from Pterolactam proved to be good candidates for the development of biosourced fungicides, with compound 3o being the most broader-spectrum agent, active against five strains and devoted of any cytotoxicity.

Full text of DOI:10.1016/j.fitote.2020.104581

Fitoterapia143(2020)104581
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Design, synthesis and antifungal activity of pterolactam-inspired amide
Mannich bases
Anca-Elena Dascalua,b,c,d, Alina Ghineta,b,d, Emmanuelle Lipkaa,c, Christophe Furmana,e
Benoit 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:
Pterolactam (5-methoxypyrrolidin-2-one) is a heterocycle naturally occurring in plants. In an attempt to identify
antifungal agents, a series of novel Mannich bases of amide derivated from Pterolactam have been designed,
synthesized and their antifungal activities were evaluated on a panel of nine fungal strains and three non albicans
candida yeasts species which have demonstrated reduced susceptibility to commonly used antifungal drugs. A
third of the target compounds exhibited good to high antifungal activities on at least one strain with EC50 lower
than the control antifungal agent. N,N′-aminals derivated from Pterolactam proved to be good candidates for the
development of biosourced fungicides, with compound 3o being the most broader-spectrum agent, active against
five strains and devoted of any cytotoxicity.
Antifungal agents
Biological screening
Pterolactam
N,N′-aminal
N,O-acetal
1. Introduction
their presence in traditional approaches towards fungal diseases [7]. It
is well known that many plant-derived metabolites exhibit antifungal
Statistics show that nearly two million people die each year from
invasive fungal infections, and that, for instance, this mortality is three-
fold higher than for malaria [1]. Synthetic drugs have proved to be
highly successful in the past, but the fungal resistance poses a real risk,
which comes naturally from the use of antifungal drugs, and many
novel resistance patterns have been already observed [2]. Despite the
numerous available antimicrobial agents the treatment of pathogenic
microbial contaminations remains a central challenge in the global
healthcare [3] and in particular the increase of fungal resistance led to
make mycoses a major cause of morbidity and mortality in im-
munocompromised patients [4]. Fungi also trigger most severe plant
pathologies and are one of the principal sources of agricultural losses
[5]. The identification of new antifungal schedule is thus imbedded in
drug discovery among the different scientific disciplines [6]. Plants
afford a valuable resource platform for such endeavors owing the
chemical diversity of the many scaffolds they provide, contributing to
properties [8–10]. Pterolactam A was extracted from bracken (Pteridium
aquilinum Kuhn var. latiusculum Underwood) [11], from the leaves of
Phyteuma japonicum [12], from Villasenoria orcuttii, the only species of
the genus Villasenoria (Asteraceae, Senecioneae), which is a shrub that
grows in the Mexican rainforest at 100 to 2000 m of elevation [13], or
from the aerial parts of Chrysanthemum coronarium L. [14] (Fig. 1).
This natural occurring 5-membered lactam ring belongs to the
pyrrolidine-2-one derivatives. It is notable that pyrrolidone derivatives
are well described for their antifungal properties [15–17]. Moreover,
N,N′-aminals and N,O-acetals originated from the substitution of the
position 5 of this heterocycle are largely distributed among the plant
metabolites.
From a chemical point of view, these scaffolds are chemically
equivalent to constrained Mannich bases of amides, many of them
known for their antifungal properties [23,24]. However, to the best of
our knowledge, no systematic study of the antifungal properties of
⁎ Corresponding author at: 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.
E-mail address: muriel.billamboz@yncrea.fr (M. Billamboz).
https://doi.org/10.1016/j.fitote.2020.104581
Received 24 February 2020; Received in revised form 26 March 2020; Accepted 26 March 2020
Availableonline29March2020
0367-326X/©2020ElsevierB.V.Allrightsreserved.

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
Fig. 1. Natural compounds derivated from pyrrolidin-2-one in position 5 [18-22, 29].
entities issued from natural Pterolactam A was reported, and only 5-
hydroxypyrrolidone B was described as antibacterial (Fig. 1). Thus, we
decided to examine a diversity of representative derivatives of these
natural building blocks, and four series of related 5-substituted 2-pyr-
rolidinones were synthesized, covering the range of aliphatic ethers and
thioethers (Series a), aliphatic amines (Series b), aromatic amines
(Series c) and heterocyclic amines (Series d) (Scheme 1).
2.2. General procedure for the preparation of target compounds
The target compounds were synthesized according to a clean solvent-
free protocol recently reported by the lab (Scheme 2) [25]. Pterolactam A
(17.4 mmol, 1 eq) and CsF (0.086 mmol, 5 mol%)were added to the nu-
cleophile (17.4 mmol, 1 eq), without any solvent, and the mixture was
stirred under moderate vacuum (30 mmHg) at 80 °C, until the 1H NMR
conversion showed no more progression, or after caking of the medium.
After cooling at room temperature, diethyl ether (20 mL) was added to the
crude mixture. The precipitate was filtered off and the solid obtained was
recrystallized from EtOH or purified by flash chromatography (silica gel,
gradient of ethyl acetate in n-heptane), to afford the target compounds. In
these conditions, N,O and N,S-acetals 1a-j, and N,N′-aminals 2a-g, 3a-t
and 4a-f were obtained in moderate to excellent yields (Table 1).
2. Material and methods
2.1. Chemistry
Solvents of analytical reagent grade were used without further
purification. Analytical thin-layer chromatographies (TLC) were per-
formed on E. Merck 60 F254 silica gel plates. 1H NMR and 13C NMR
spectra were obtained at 25 °C on a Varian 400-MR spectrometer
(400 MHz for 1H, 100 MHz for 13C, respectively). Chemical shifts (δ) are
quoted in parts per million (ppm) and are referenced to TMS as an
internal standard. Coupling constants (J) are quoted in hertz. Column
chromatographies were performed with a CombiFlash Rf Companion
(Teledyne-Isco System) using RediSep packed columns. IR spectra were
recorded on a Varian 640-IR FT-IR Spectrometer. Melting points were
measured on a MPA 100 OptiMelt® apparatus. Elemental analyses (C,
H, N, S) of new compounds were determined on a Thermo Electron
apparatus by ‘Pôle Chimie Moléculaire-Welience’, Faculté de Sciences
Mirande, Université de Bourgogne, Dijon, France. Yield refers to the
isolated analytically pure material. Starting materials were purchased
from Solabia group (France), Sigma Aldrich, Alfa Aesar and TCI, and
were used without further purification.
2.2.1. 5-Phenethoxypyrrolidin-2-one (1a)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 1a as a white solid (0.25 g, 12%
yield); m.p. 144–145 °C; 1H-NMR (400 MHz, DMSO-d6): δ 8.66 (s, 1H,
NH), 7.30–7.19 (m, 5H, CHAr), 4.89 (d, J = 6.5 Hz, 1H), 3.65 (q,
J = 7.0 Hz, 1H, CH2-CH2-O), 3.49 (q, J = 7.0 Hz, 1H, CH2-CH2-O),
2.79 (t, J = 7.9 Hz, 2H, CH2-CH2-O), 2.28–2.12 (m, 2H, CH2-CH2-CH),
2.06–1.96 (m, 1H, CH2-CH2-CH), 1.87–1.78 (m, 1H, CH2-CH2-CH); 13C-
NMR (100 MHz, DMSO-d6): δ 177.9 (C=O), 139.2 (C), 129.3 (2 CHAr),
128.6 (2 CHAr), 126.5 (CHAr), 85.6 (CH), 67.7 (CH2), 35.9 (CH2), 28.5
(CH2), 28.0 (CH2). IR ν (cm−1): 3178, 3106, 2913, 1683, 1278, 1067,
1027. Anal. Calcd for C12H15NO2: C, 70.22; H, 7.37; N, 6.82; found: C,
69.84; H, 7.51; N, 7.03%.
Scheme 1. Fungicidal Mannich bases of amides (1–4) from Pterolactam A.
2

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
Scheme 2. Reagents and conditions: 1 eq of H2N-NH-R, CsF (5 mol%), solvent-less, 6–20 h, 80 °C [25]
2.2.2. 5-((4-Hydroxybenzyl)oxy)pyrrolidin-2-one (1b)
2.2.3. 5-((4-Methoxybenzyl)oxy)pyrrolidin-2-one (1c)
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of n-
heptane/ethyl acetate, the wanted compound being eluted in 70% ethyl
acetate, as a white solid (1.2 g, 33% yield); m.p. 142–143 °C; 1H-NMR
(400 MHz, DMSO-d6): δ 9.38 (s, 1H, OH), 8.76 (s, 1H, NH), 7.12 (d,
J = 8.5 Hz, 2H, CHAr), 6.73 (d, J = 8.5 Hz, 2H, CHAr), 4.93 (d,
J = 6.2 Hz, 1H, CH), 4.39 (d, J = 11.4 Hz, 1H, CH2-O), 4.25 (d,
J = 11.4 Hz, 1H, CH2-O), 2.29–2.13 (m, 2H, CH2-CH2-CH), 2.06–1.98
(m, 1H, CH2-CH2-CH), 1.87–1.82 (m, 1H, CH2-CH2-CH); 13C-NMR
(100 MHz, DMSO-d6): δ 178.0 (C=O), 157.24 (C), 130.0 (2 CHAr),
128.6 (C), 115.4 (2 CHAr), 84.8 (CH), 68.3 (CH2), 28.5 (CH2), 28.0
(CH2). IR ν (cm−1): 3203, 2935, 1668, 1516, 1454, 1227, 1056, 1037.
Anal. Calcd for C11H13NO3: C, 63.76; H, 6.32; N, 6.76; found: C, 63.36;
H, 6.40; N, 6.91%.
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of n-
heptane/ethyl acetate, the wanted compound being eluted in 70% ethyl
acetate, as a of a white solid (1.2 g, 46% yield); m.p. 71–72 °C; 1H-NMR
(400 MHz, DMSO-d6): δ 8.78 (s, 1H, NH), 7.25 (d, J = 6.9 Hz, 2H,
CHAr), 6.90 (d, J = 6.9 Hz, 2H, CHAr), 4.94 (d, J = 6.5 Hz, 1H), 4.45
(d, J = 11.6 Hz, 1H, CH2), 4.30 (d, J = 11.6 Hz, 1H, CH2), 3.73 (s, 3H,
OCH3), 2.32–2.14 (m, 2H, CH2-CH2-CH), 2.03–2.01 (m, 1H, CH2-CH2-
CH), 1.88–1.84 (m, 1H, CH2-CH2-CH); 13C-NMR (100 MHz, DMSO-d6):
δ 178.0 (C=O), 159.1 (C), 130.4 (C), 129.8 (2 CHAr), 114.1 (2 CHAr),
84.9 (CH), 68.0 (CH2), 55.5 (CH3), 28.5 (CH2), 28.0 (CH2). IR ν (cm−1):
3413, 3194, 2914, 1668, 1256, 1172, 1027. Anal. Calcd for C12H15NO3:
C, 65.14; H, 6.83; N, 6.33; found: C, 64.95; H, 6.91; N, 6.07%.
3

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Fitoterapia143(2020)104581
Table 1
Antifungal activity of target compounds at 100 μg/mL concentration.a,b
SS - Sclerotinia sclerotiorum; BC - Botrytis cinerea; AO - Aspergillus oryzae; PV - Paecilomyces variotii; PO - Penicillium ochrochloron; CC - Cladosporium cladosporioides; AA -
Alternaria alternata; FS - Fusarium solani; GC - Geotrichum candidum; CK - Candida krusei; CP - Candida pseudotropicalis; CT - Candida tropicalis.
a
b
All the data are the average value of three replications.
The colored bold values represent data above or equal to 75% inhibition of the corresponding strain.
2.2.4. 5-((3-Hydroxy-4-methoxybenzyl)oxy)pyrrolidin-2-one (1d)
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of n-
heptane/ethyl acetate, the wanted compound being eluted in 70% ethyl
acetate, as a white solid (1.2 g, 30% yield); m.p. 121–122 °C; 1H-NMR
(400 MHz, DMSO-d6): δ 8.93 (s, 1H, OH), 8.73 (bs, 1H, NH), 6.85 (d,
J = 8.4 Hz, 1H, CHAr), 6.75 (d, J = 1.8 Hz, 1H, CHAr), 6.71 (dd,
J = 8.4 Hz, J = 1.8 Hz, 1H, CHAr), 4.94 (d, J = 6.2 Hz, 1H, CH), 4.39
(d, J = 11.7 Hz, 1H, CH2), 4.24 (d, J = 11.7 Hz, 1H, CH2), 3.74 (s, 3H,
CH3), 2.33–2.13 (m, 2H, CH2-CH2-CH), 2.08–1.98 (m, 1H, CH2-CH2-
4

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
for C17H15Cl2NO2: C, 60.73; H, 4.50; N, 4.17; found: C, 60.53; H, 4.59;
CH), 1.92–1.83 (m, 1H, CH2-CH2-CH); 13C-NMR (100 MHz, DMSO-d6):
δ 178.0 (C=O), 147.5 (C), 146.8 (C), 131.0 (C), 119.1 (CHAr), 115.7
(CHAr), 112.3 (CHAr), 84.9 (CH), 68.3 (CH2), 56.1 (CH3), 28.5 (CH2),
28.0 (CH2). IR ν (cm−1): 3212, 3194, 1674, 1679, 1254, 1172, 1027.
Anal. Calcd for C12H15NO4: C, 60.75; H, 6.37; N, 5.90; found: C, 60.36;
H, 6.40; N, 6.12%.
N, 4.23%.
2.2.9. 5-(Phenylthio)pyrrolidin-2-one (1j) [27]
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the compound as a white solid (1.0 g, 30%
yield); m.p.75–76 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.48 (s, 1H, NH),
7.47 (dd, J = 7.9 Hz, J = 2.1 Hz, 2H, CHAr), 7.38–7.33 (m, 1H, CHAr),
7.35 (dd, J = 7.9 Hz, J = 2.1 Hz, 2H, CHAr), 5.16 (d, J = 7.4 Hz, 1H,
CH), 2.46–2.43 (m, 1H, CH2CH2CH), 2.03–1.80 (m, 3H, CH2CH2CH);
13C{1H}NMR (DMSO-d6, 100 MHz): δ 176.8 (C=O), 133.3 (2 CHAr),
132.9 (C), 129.6 (2 CHAr), 128.2 (CHAr), 62.2 (CH), 29.2 (CH2), 28.9
(CH2). IR ν (cm−1): 3389, 3158, 1657, 1453, 1258.
2.2.5. 5-((3,4,5-Trimethoxybenzyl)oxy)pyrrolidin-2-one (1e)
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of n-
heptane/ethyl acetate, the wanted compound being eluted in 70% ethyl
acetate, being obtained as a white solid (1.8 g, 40% yield); m.p.
91–92 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.76 (s, 1H, NH), 6.63 (s,
2H, CHAr), 4.96 (d, J = 5.2 Hz, 1H, CH), 4.46 (d, J = 9.6 Hz, 1H, CH2),
4.32 (d, J = 9.6 Hz, 1H, CH2), 3.77 (s, 6H, OCH3), 3.64 (s, 3H, OCH3),
2.32–2.28 (m, 2H, CH2CH2CH), 2.10–2.03 (m, 1H, CH2CH2CH),
1.98–1.92 (m, 1H, CH2CH2CH); 13C{1H}NMR (DMSO-d6, 100 MHz): δ
178.0 (C=O), 153.2 (2 C), 137.2 (C), 134.1 (C), 105.4 (2 CHAr), 85.0
(CH), 68.5 (CH2), 60.4 (OCH3), 56.2 (2 OCH3), 28.5 (CH2), 28.0 (CH2).
IR ν (cm−1): 3184, 3092, 1682, 1590, 1462, 1230, 1132. Anal. Calcd
for C14H19NO5: C, 59.78; H, 6.81; N, 4.98; found: C, 60.01; H, 6.99; N,
5.03%.
2.2.10. 5-((2-Hydroxyethyl)amino)pyrrolidin-2-one (2a)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 2a as a white solid (1.75 g, 70%
yield); m.p. 136–137 °C; 1H NMR (DMSO-d6, 400 MHz): δ ppm 8.09 (bs,
1H, NH), 4.51 (bs, 1H, CH), 4.34 (bs, 1H, NH), 3.41 (bs, 2H, CH2), 2.67
(bs, 1H, OH), 2.15–2.01 (m, 5H, CH2CH2NH, CH2CH2CH), 1.65–1.60
(m, 1H, CH2CH2CH); 13C{1H}NMR (DMSO-d6, 100 MHz): δ ppm 176.8
(C=O), 69.7 (CH), 61.3 (CH2), 48.0 (CH2), 47.5 (CH2), 29.7 (CH2), 28.3
(CH2). IR ν (cm−1): 3223, 3079, 2850, 1668, 1455, 1261, 1060.
2.2.6. 5-(Benzo[d][1,3]dioxol − 5-ylmethoxy)pyrrolidin-2-one (1f)
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of
DCM/methanol, the wanted compound being eluted in 20% methanol,
affording the compound as a white solid (0.4 g, 10% yield); m.p.
134–135 °C; 1H NMR (400 MHz, CDCl3): δ 8.0 (s, 1H, NH), 6.83 (s, 1H,
CHAr), 6.78 (s, 2H, CHAr), 5.95–5.91 (m, 2H, CH2), 5.05 (d,
J = 6.0 Hz, 1H, CH), 4.50 (d, J = 11.2 Hz, 1H, CH2), 4.38 (d,
J = 11.2 Hz, 1H, CH2), 2.57–2.32 (m, 1H, CH2CH2CH), 2.30–2.22 (m,
2H, CH2CH2CH), 2.21–2.08 (m, 1H, CH2CH2CH); 13C{1H}NMR
(100 MHz, CDCl3): δ 179.6 (C=O), 147.8 (C), 147.3 (CIV), 131.1 (C),
121.5 (CHAr), 108.6 (CHAr), 108.2 (CHAr), 101.0 (CH2), 85.2 (CH),
69.4 (CH2), 28.4 (CH2), 28.3 (CH2). IR ν (cm−1): 3205, 3156, 1684,
1252, 1101, 1078. Anal. Calcd for C12H13NO4: C, 61.27; H, 5.57; N,
5.95; found: C, 61.42, H, 5.59; N, 5.97%.
2.2.11. 5-Morpholinopyrrolidin-2-one (2b) [28]
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the compound as a white-off solid (2.0 g, 70%
yield); m.p. 141–142 °C; 1H-NMR (400 MHz, CDCl3): δ 7.29 (bs, 1H,
NH), 4.48 (d, J = 7.4 Hz, 1H, CH), 3.73 (d, J = 5.2 Hz, 4H, CH2), 2.38
(d, J = 5.2 Hz, 4H, CH2), 2.30–2.26 (m, 3H, CH2CH2CH), 2.08–2.03 (m,
1H, CH2CH2CH); 13C-NMR (100 MHz, CDCl3): δ 178.4 (C=O), 75.1
(CH), 66.8 (2 CH2), 47.3 (2 CH2), 29.4 (CH2), 23.1 (CH2). IR ν (cm−1):
3153, 2966, 2854, 1673, 1263, 1107, 1068.
2.2.12. 5-(4-Phenylpiperazin-1-yl)pyrrolidin-2-one (2c)[27]
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 2c as a white solid (4.0 g, 95%
yield); m.p. 174–175 °C; 1H NMR (DMSO-d6, 400 MHz): δ 7.22 (d,
J = 8.0 Hz, 2H, CHAr), 6.92 (d, J = 7.8 Hz, 2H, CHAr), 6.76 (t,
J = 7.6 Hz, 1H, CHAr), 4.41 (dd, J = 7.9 Hz, J = 2.25 Hz, 1H, CH),
3.11 (t, J = 5.0 Hz, 4H, NCH2CH2N), 2.70–2.60 (m, 4H, NCH2CH2N),
2.22–2.08 (m, 3H, CH2CH2CH), 1.86–1.82 (m, 1H, CH2CH2CH);
13C{1H}NMR (DMSO-d6, 100 MHz): δ 177.2 (C=O), 151.4 (C), 129.3
(2 CHAr), 119.2 (CHAr), 115.8 (2 CHAr), 74.4 (CH), 48.6 (2 CH2), 46.8
(2 CH2), 26.6 (CH2), 23.7 (CH2). IR ν (cm−1): 3214, 2825, 1686, 1651,
1241.
2.2.7. 5-(Benzhydryloxy)pyrrolidin-2-one (1 h)
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of n-
heptane/ethyl acetate, the wanted compound being eluted in 90% ethyl
acetate, as a white solid (1.1 g, 24% yield); m.p. 98–99 °C; 1H NMR
(DMSO-d6, 400 MHz): δ 8.79 (s, 1H, NH) 7.29–7.36 (m, 10H, ArH), 5.63
(s, 1H, CH), 4.85 (d, J = 5.8 Hz, 1H, CH), 2.35–2.25 (m, 1H,
CH2CH2CH), 2.17–2.13 (m, 1H, CH2CH2CH), 2.10–1.85 (m, 2H,
CH2CH2CH); 13C{1H}NMR (DMSO-d6, 100 MHz): δ 178.0 (C=O), 143.0
(C), 142.3 (C), 128.9 (2 CHAr), 128.7 (2 CHAr), 127.9 (CHAr), 127.6
(CHAr), 127.5 (2 CHAr), 126.8 (2 CHAr), 85.7 (CH), 78.7 (CH), 28.5
(CH2), 28.2 (CH2). IR ν (cm−1): 3183, 3104, 2935, 1697, 1283, 1059.
Anal. Calcd for C17H17NO2: C, 76.38; H, 6.41; N, 5.24; found: C, 76.12;
H, 6.26; N, 5.08%.
2.2.13. 5-(4-(4-Fluorophenyl)piperazin-1-yl)pyrrolidin-2-one (2d)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (50 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the compound as a white solid (3.6 g, 78%
yield); m.p. 172–173 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.23 (bs, 1H,
NH), 7.04 (t, J = 9.2 Hz, 2H, ArH), 6.95 (dd, J = 8.8 Hz, J = 4.8 Hz,
2H, CHAr), 4.41 (d, J = 5.2 Hz, 1H, CH), 3.06 (bs, 4H, NCH2CH2-N),
2.68–2.65 (m, 2H, NCH2CH2N), 2.50–2.46 (m, 2H, NCH2CH2N),
2.20–2.08 (m, 3H, CH2CH2CH), 1.89–1.87 (m, 1H, CH2CH2CH);
13C{1H}NMR (DMSO-d6, 100 MHz): δ 177.3 (C=O), 157.6 (C, JC-
F = 233.9 Hz), 148.3 (C, JC-F = 1.5 Hz), 117.5 (2 CHAr, JC-F = 7.6 Hz),
115.6 (2 CHAr, JC-F = 21.3 Hz), 74.4 (CH), 49.4 (2 CH2), 46.8 (2 CH2),
29.7 (CH2), 23.8 (CH2). IR ν (cm−1): 3214, 2825, 1686, 1651, 1241.
Anal. Calcd for C14H18FN3O: C, 63.86; H, 6.89; N, 15.96; found: C,
64.01; H, 6.59; N, 15.73%.
2.2.8. 5-(Bis(4-chlorophenyl)methoxy)pyrrolidin-2-one (1i)
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of n-
heptane/ethyl acetate, the wanted compound being eluted in 80% ethyl
acetate, as a white solid (0.6 g, 10% yield); m.p. 172–173 °C; 1H-NMR
(DMSO-d6, 400 MHz): δ ppm 8.79 (s, 1H, NH), 7.44–7.30 (m, 8H,
CHAr), 5.65 (s, 1H, NH), 4.85 (d, 1H, J = 6.4 Hz, CH), 2.41–2.27 (m,
1H, CH2CH2CH), 2.21–2.18 (m, 1H, CH2CH2CH), 2.05–1.95 (m, 2H,
CH2CH2CH); 13C-NMR (100 MHz, DMSO-d6): δ 178.0 (C), 141.6 (C),
140.9 (C), 132.7 (C), 132.3 (C), 129.4 (2 CHAr), 129.0 (2 CHAr), 128.8
(2 CHAr), 128.7 (2 CHAr), 83.9 (C), 77.2 (CH), 28.4 (CH2), 28.2 (CH2).
IR ν (cm−1): 3196, 3160, 2915, 1695, 1488, 1278, 1045. Anal. Calcd
5

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
2.2.14. 5-(Phenethylamino)pyrrolidin-2-one (2e)
2.2.19. 5-((4-Hydroxyphenyl)amino)pyrrolidin-2-one (3c)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 2e as a white solid (2.4 g, 68%
yield); m.p. 71–72 °C; 1H-NMR (400 MHz, DMSO-d6): δ 8.15 (bs, 1H,
NH), 7.26 (d, J = 7.9 Hz, 2H, CHAr), 7.19 (d, J = 7.9 Hz, 2H, CHAr),
7.16 (d, J = 7.9 Hz, 1H, CHAr), 4.34 (s, 1H, CH), 2.86–2.84 (m, 1H,
NH), 2.68–2.64 (m, 3H, CH2-CH2), 2.19–2.14 (m, 3H, CH2-CH2),
2.03–1.90 (m, 1H, CH2-CH2), 1.66–1.58 (m, 1H, CH2-CH2-CH); 13C-
NMR (100 MHz, DMSO-d6): δ 176.74 (C=O), 140.84 (C), 129.04 (2
CHAr), 128.63 (2 CHAr), 126.27 (CHAr), 69.50 (CH), 47.45 (CH2),
36.58 (CH2), 29.67 (CH2), 28.3 (CH2). IR ν (cm−1): 3301, 3286, 3173,
2943, 1681, 1497. Anal. Calcd for C12H16N2O: C, 70.56; H, 7.90; N,
13.71; found: C, 70.53; H, 7.59; N, 13.73%.
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 3c as a white solid (1.8 g, 50%
yield); m.p. 190–191 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.49 (s, 1H,
NH), 8.16 (s, 1H, NH), 6.48 (d, J = 9.1 Hz, J = 2.46 Hz, 2H, CHAr),
6.55 (d, J = 9.1 Hz, J = 2.46 Hz, 2H, CHAr), 5.47 (d, J = 9.5 Hz, 1H,
CH), 4.99–4.97 (m, 1H, OH), 2.31–2.26 (m, 2H, CH2CH2CH), 2.12–2.04
(m, 1H, CH2CH2CH), 1.82–1.74 (m, 1H, CH2CH2CH); 13C{1H}NMR
(DMSO-d6, 100 MHz): δ 176.74 (C=O), 149.50 (C), 139.76 (C), 116.11
(2 CHAr), 115.12 (2 CHAr), 65.45 (CH), 29.56 (CH2), 28.47 (CH2). IR ν
(cm−1): 3361, 3141, 1658, 1511, 1244, 1231. Anal. Calcd for
C
10H12N2O2: C, 62.49; H, 6.29; N, 14.57; found: C, 62.49; H, 6.14; N,
14.20%.
2.2.20. 5-((2,4-Dimethoxyphenyl)amino)pyrrolidin-2-one (3d)
2.2.15. 5-(Benzylamino)pyrrolidin-2-one (2f) [28]
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 3d as a white solid (3.5 g, 85%
yield); m.p. 135–136 °C; 1H NMR (DMSO-d6, 400 MHz): δ 6.59 (d,
J = 8.2 Hz, 1H, CHAr), 6.47 (d, J = 2.4 Hz, 1H, CHAr), 6.44 (dd,
J = 8.2 Hz, J = 2.4 Hz, 1H, CHAr), 6.32 (bs, 1H, NH), 5.22 (dd,
J = 6.7 Hz, J = 4.6 Hz, 1H, CH), 4.16 (bs, 1H, NH), 3.82 (s, 3H, CH3),
3.76 (s, 3H, CH3), 2.65–2.35 (m, 3H, CH2CH2CH), 2.02–1.93 (m, 1H,
CH2CH2CH); 13C{1H}NMR (DMSO-d6, 100 MHz): δ 176.8 (C=O),
153.6 (C), 148.9 (C), 128.9 (C), 112.7 (CHAr), 103.9 (CHAr), 99.5
(CHAr), 65.7 (CH), 55.7 (CH3), 55.5 (CH3), 29.2 (CH2), 28.8 (CH2).
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 2f as a white solid (2.6 g, 79% yield);
m.p. 106–107 °C; 1H NMR (DMSO-d6, 400 MHz): δ 7.34–7.27 (m, 5H,
CHAr), 4.62 (d, J = 6.3 Hz, 1H, CH), 3.87 (d, J = 12.7 Hz, 1H, CH2-
NH), 3.82 (d,
J = 12.7 Hz, 1H, CH2-NH), 2.38–2.46 (m, 2H,
CH2CH2CH), 1.85–1.80 (m, 1H, CH2CH2CH); 13C{1H}NMR (DMSO-d6,
100 MHz): δ 177.8 (C=O), 139.5 (C), 128.6 (2 CHAr), 128.1 (2 CHAr),
121.3 (CHAr), 69.5 (CH), 50.0 (CH2), 29.5 (CH2), 28.7 (CH2). IR ν
(cm−1): 3264, 3029, 1677, 1487, 1262.
2.2.16. 5-((4-Chlorobenzyl)amino)pyrrolidin-2-one (2 g)
2.2.21. 5-((2-Chlorophenyl)amino)pyrrolidin-2-one (3e)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 2 g as a white solid (3.2 g, 81%
yield); m.p. 145–146 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.23 (bs, 1H,
NH), 7.33 (s, 4H, CHAr), 4.29 (bs, 1H, CH), 3.75 (d, J = 14.2 Hz, 1H,
CH2), 3.59 (d, J = 14.2 Hz, 1H, CH2), 2.75 (bs, 1H, NH), 2.25–2.10 (m,
2H, CH2CH2CH), 2.03–1.96 (m, 1H, CH2CH2CH), 1.69–1.62 (m, 1H,
CH2CH2CH); 13C{1H}NMR (DMSO-d6, 100 MHz): δ 176.8 (C=O),
140.2 (C), 131.4 (C), 130.2 (2 CHAr), 128.4 (2 CHAr), 68.9 (CH), 48.4
(CH2), 29.7 (CH2), 28.2 (CH2). Anal. Calcd for C10H12N2O: C, 58.80; H,
5.83; N, 12.47; found: C, 58.76; H, 5.76; N, 12.63%.
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the wanted compound as a white solid (3.1 g,
85% yield); m.p. 125–126 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.32 (s,
1H, NH), 7.28 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H, CHAr), 7.14 (td,
J = 8.8 Hz, J = 2.0 Hz, 1H, CHAr), 6.84 (d, J = 7.2 Hz, 1H, CHAr),
6.66 (td, J = 8.0 Hz, J = 1.6 Hz, 1H, CHAr), 5.61 (d, J = 8.8 Hz, 1H,
NH), 5.22 (t, J = 4.0 Hz, 1H, CH), 2.40–2.25 (m, 2H, CH2CH2CH),
2.13–2.06 (m, 2H, CH2CH2CH); 13C{1H}NMR (DMSO-d6, 100 MHz): δ
176.9 (C=O), 142.7 (C), 129.6 (C), 128.4 (CHAr), 118.9 (C), 118.3
(CH), 113.3 (CHAr), 64.2 (CH), 29.5 (CH2), 28.1 (CH2); IR ν (cm−1):
3398, 3169, 1692, 1597, 1497, 1265, 1181; Anal. Calcd for
C10H11ClN2O: C, 57.01; H, 5.26; N, 13.30%.
2.2.17. 5-(Phenylamino)pyrrolidin-2-one (3a) [30]
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 3a as a white solid (2.7 g, 88%
yield); m.p. 135–136 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.25 (bs, 1H,
NH), 7.09 (t, J = 6 Hz, 2H, CHAr), 6.62 (d, J = 9.6 Hz, 2H, CHAr), 6.57
(d, J = 6.6 Hz, 1H, NH), 6.11 (d, J = 9.6 Hz, 1H, CHAr), 5.10 (bs, 1H,
CH), 2.46–2.20 (m, 2H, CH2CH2CH), 2.16–2.05 (m, 1H, CH2CH2CH),
1.95–1.73 (m, 1H, CH2CH2CH); 13C{1H}NMR (DMSO-d6, 100 MHz): δ
176.8 (C=O), 147.2 (C), 129.4 (2 CHAr), 117.1 (CHAr), 114.4 (2
CHAr), 64.2 (CH), 29.5 (CH2), 28.5 (CH2). IR ν (cm−1): 3338, 3165,
1669, 1598, 1253.
2.2.22. 5-((3-Chlorophenyl)amino)pyrrolidin-2-one (3f) [30]
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the wanted compound as a white solid (2.8 g,
77% yield); m.p. 139–140 °C; 1H NMR (DMSO-d6, 400 MHz): δ 7.10 (t,
J = 7.9 Hz, 1H, CHAr), 6.66 (t, J = 2.2 Hz, 1H, CHAr), 6.57 (td,
J = 7.9 Hz, J = 2.2 Hz, 2H, CHAr), 5.10 (t, J = 5.2 Hz, 1H, CH),
2.37–2.25 (m, 2H, CH2CH2CH), 2.12–2.06 (m, 1H, CH2CH2CH),
1.82–1.79 (m, 1H, CH2CH2CH); 13C{1H}NMR (DMSO-d6, 100 MHz): δ
176.8 (C=O), 148.8 (C), 134.1 (C), 130.8 (CHAr), 116.4 (CHAr), 112.5
(CHAr), 112.0 (CHAr), 64.0 (CH), 29.5 (CH2), 28.4 (CH2);
2.2.18. 5-(p-Tolylamino)pyrrolidin-2-one (3b) [30]
IR ν (cm−1): 3328, 3168, 1682, 1595, 1482, 1242, 1089.
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (30 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 3b as a white solid (2.8 g, 85%
yield); m.p. 139–140 °C; 1H-NMR (DMSO-d6, 400 MHz): δ ppm 6.90 (d,
2H, J = 8.2 Hz, CHAr) 6.54 (d, 2H, J = 8.2 Hz, CHAr), 5.93 (d, 1H,
J = 8.2 Hz, NH), 5.05 (td, 1H, J = 8.2 Hz, J = 4.1 Hz, CH), 2.46–2.29
(m, 2H, CH2CH2CH), 2.15 (s, 3H, CH3), 2.09–2.13 (m, 1H, CH2CH2CH),
1.85–1.78 (m, 1H, CH2CH2CH); 13C-NMR (100 MHz, DMSO-d6): δ ppm
176.8 (C), 144.9 (C), 129.8 (2 CHAr), 125.5 (C), 113.6 (2 CHAr), 64.5
(CH), 29.5 (CH2), 28.4 (CH2), 20.5 (CH3). IR ν (cm−1): 3283, 3161,
1680, 1583, 1485, 1246, 1176.
2.2.23. 5-((4-Chlorophenyl)amino)pyrrolidin-2-one (3 g)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the wanted compound as a grey solid (2.1 g,
57% yield); m.p. 169–170 °C; 1H-NMR (DMSO-d6, 400 MHz): δ ppm
7.12 (d, J = 9.2 Hz, 2H, CHAr) 6.63 (d, J = 9.2 Hz, 2H, CHAr), 6.39
(bs, 1H, NH), 5.08 (s, 1H, CH), 2.25–2.31 (m, 2H, CH2CH2CH),
2.07–2.12 (m, 1H, CH2CH2CH), 1.84–1.79 (m, 1H, CH2CH2CH); 13C-
NMR (100 MHz, DMSO-d6) δ ppm 176.8 (C) 146.2 (C), 129.0 (2 CHAr),
120.3 (C), 114.8 (2 CHAr), 64.2 (CH), 29.5 (CH2), 28.4 (CH2). IR ν
6

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
(cm−1): 3281, 3159, 3088, 1681, 1590, 1487, 1247, 1176. Anal. Calcd
for C10H11ClN2O: C, 57.01; H, 5.26; N, 13.30; found: C, 61.55; H, 5.61;
N, 14.22%.
2.17–2.23 (m, 1H, CH2CH2CH), 1.90–1.84 (m, 1H, CH2CH2CH); 13C-
NMR (DMSO-d6, 100 MHz): δ ppm 176.8 (C=O), 154.0 (C), 126.1 (2
CHAr), 112.6 (2 CHAr), 68.3 (CH), 30.5 (CH2), 29.6 (CH2), 25.1 (CH3).
IR ν (cm−1): 3151, 3066, 2892, 1693, 1583, 1462, 1311, 1238, 1081.
2.2.24. 5-((4-Fluorophenyl)amino)pyrrolidin-2-one (3 h)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 3 h as a white solid (2.8 g, 82%
yield); m.p. 146–147 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.15 (bs, 1H,
NH), 6.93 (t, J = 8.8 Hz, 2H, CHAr), 6.63 (dd, J = 8.8 Hz, J = 4.4 Hz,
2H, CHAr), 6.11 (d, J = 7.8 Hz, 1H, NH), 5.05 (bs, 1H, CH), 2.36–2.26
(m, 2H, CH2CH2CH), 2.14–2.05 (m, 1H, CH2CH2CH), 1.85–1.80 (m, 1H,
CH2CH2CH); 13C{1H}NMR (DMSO-d6, 100 MHz): δ 176.85 (C=O),
155.2 (C, C-F, J = 232 Hz), 143.85 (C), 115.7 (2 CHAr, J = 21.2 Hz),
114.3 (2 CHAr, J = 7.1 Hz), 64.7 (CH), 29.5 (CH2), 28.4 (CH2). IR ν
(cm−1): 3284, 3177, 1683, 1505, 1245. Anal. Calcd for C10H11FN2O: C,
61.85; H, 5.71; N, 14.42; found: C, 61.55; H, 5.61; N, 14.22%.
2.2.29. 5-((2-Hydroxy-4-nitrophenyl)amino)pyrrolidin-2-one (3 m)
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of n-
heptane/ethyl acetate, the wanted compound being eluted in 100%
ethyl acetate, as 4.0 g of a yellow solid, in 55% yield; m.p. 169–170 °C;
1H-NMR (DMSO-d6, 400 MHz): δ ppm 10.42 (bs, 1H, OH), 8.34 (s, 1H,
NH), 7.68 (d, J = 9.0 Hz, 1H, CHAr), 7.51 (d, J = 2.73 Hz, 1H, CHAr),
6.74 (d, J = 9.0 Hz, 1H, CHAr), 6.51 (d, J = 9.0 Hz, 1H, NH), 5.32 (td,
J = 8.5, 3.8 Hz, 1H, CH), 2.35–2.39 (m, 2H, CH2CH2CH), 2.04–2.11
(m, 2H, CH2CH2CH); 13C-NMR (DMSO-d6, 100 MHz): δ ppm 177.0
(C=O), 143.7 (C), 143.0 (C), 136.9 (C), 118.2 (CHAr), 109.0 (CHAr),
108.3 (CHAr), 63.6 (CH), 29.4 (CH2), 27.9 (CH2). IR ν (cm−1): 3404,
3332, 1658, 1589, 1475, 1255, 1221.
2.2.25. 5-((2-Nitrophenyl)amino)pyrrolidin-2-one (3i)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the compound as a yellow solid (1.2 g, 30%
yield); m.p. 182–183 °C; 1H-NMR (DMSO-d6, 400 MHz): δ ppm 8.52 (s,
1H, NH), 8.09 (dd, J = 9.2, 1.6 Hz, 1H, CHAr), 7.97 (d, J = 7.5 Hz, 1H,
NH), 7.57 (t, J = 9.2 Hz, 1H, CHAr), 7.12 (d, J = 9.2 Hz, 1H, CHAr),
6.80 (d, J = 7.5 Hz, 1H, NH), 5.43 (td, J = 7.5, 3.1 Hz, 1H, CH),
2.42–2.30 (m, 2H, CH2CH2CH), 2.20–1.25 (m, 1H, CH2CH2CH),
1.90–1.86 (m, 1H, CH2CH2CH); 13C-NMR (DMSO-d6, 100 MHz): δ ppm
177.0 (C=O), 143.4 (C), 137.0 (CHAr), 132.3 (C), 126.7 (CHAr), 117.0
(CHAr), 115.8 (CHAr), 64.0 (CH), 29.2 (CH2), 28.5 (CH2). IR ν (cm−1):
3345, 3156, 1686, 1342, 1228, 1178.
2.2.30. 5-Nitro-2-((5-oxopyrrolidin-2-yl)amino)benzonitrile (3n) [30]
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of n-
heptane/ethyl acetate, the wanted compound being eluted in 100%
ethyl acetate, as a yellow solid (1.5 g, 35% yield); m.p. 199–200 °C; 1H-
NMR (DMSO-d6, 400 MHz): δ ppm 8.47 (d, J = 2.8 Hz, 1H, NH), 8.39
(d, J = 2.8 Hz, 1H, NH), 8.22 (dd, J = 10.0 Hz, J = 2.8 Hz, 1H, CHAr),
7.89 (d, J = 8.4 Hz, 1H, CHAr), 7.04 (d, J = 10 Hz, 1H, CHAr), 5.45
(td, J = 7.6, 2.5 Hz, 1H, CH), 2.43–2.40 (m, 2H, CH2CH2CH),
2.12–1.98 (m, 2H, CH2CH2CH); 13C-NMR (DMSO-d6, 100 MHz): δ ppm
177.2 (C=O), 153.1 (C), 137.1 (C), 131.4 (CHAr), 130.0 (CHAr), 116.4
(C), 113.0 (CHAr), 95.3 (C), 64.4 (CH), 29.2 (CH2), 27.6 (CH2). IR ν
(cm−1): 3332, 3167, 3094, 2230, 1697, 1587, 1503, 1305, 1270, 1171.
2.2.26. 5-((3-Nitrophenyl)amino)pyrrolidin-2-one (3j)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the compound as a yellow solid (1.9 g, 50%
yield); m.p. 170–171 °C; 1H-NMR (DMSO-d6, 400 MHz): δ ppm 8.39 (s,
1H, NH), 7.43 (d, J = 8.9 Hz, 2H, CHAr), 7.38 (t, J = 8.9 Hz, 1H,
CHAr), 7.05 (d, J = 8.9 Hz, 1H, CHAr), 6.88 (d, J = 8.4 Hz, 1H, NH),
5.23 (td, J = 7.7, 3.7 Hz, 1H, CH), 2.49–2.31 (m, 2H, CH2CH2CH),
2.17–2.10 (m, 1H, CH2CH2CH), 1.88–1.82 (m, 1H, CH2CH2CH); 13C-
NMR (100 MHz, DMSO-d6) δ ppm 176.2 (C=O), 148.7 (C), 147.8 (C),
130.0 (CHAr), 119.0 (CHAr), 110.8 (CHAr), 106.9 (CHAr), 63.4 (CH),
28.9 (CH2), 27.8 (CH2). IR ν (cm−1): 3349, 3259, 1681, 1594, 1477,
1326, 1254, 1176, 1121.
2.2.31. 5-((3-Methoxy-5-nitrophenyl)amino)pyrrolidin-2-one (3o)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the wanted compound as an orange solid (2.4 g,
55% yield); m.p. 194–195 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.53 (s,
1H, NH), 7.87 (d, J = 7.5 Hz, 1H, NH), 7.53 (s, 1H, CHAr), 7.31 (dd,
J = 9.5 Hz, J = 2.5 Hz, 1H, CHAr), 7.12 (d, J = 7.5 Hz, 1H, CHAr),
5.42 (bs, 1H, CH), 3.76 (s, 3H, CH3), 2.43–2.32 (m, 2H, CH2CH2CH),
2.22–2.11 (m, 1H, CH2CH2CH), 2.02–1.90 (m, 1H, CH2CH2CH) ppm;
13C{1H}NMR (DMSO-d6, 100 MHz): δ 176.97 (C=O), 150.27 (C),
138.99 (C), 131.58 (C), 127.16 (CHAr), 117.46 (CHAr), 107.25 (CHAr),
64.16 (CH), 56.08 (CH3.), 29.22 (CH2), 28.63 (CH2). IR ν (cm−1): 3359,
3169, 1695, 1511, 1345, 1232, 1178. Anal. Calcd for C11H13N3O4: C,
52.59; H, 5.22; N, 16.73; found: C, 52.24; H, 4.89; N, 16.36%.
2.2.27. 5-((4-Nitrophenyl)amino)pyrrolidin-2-one (3 k) [30]
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the compound as a yellow solid (2.3 g, 60%
yield); m.p. 200–201 °C; 1H-NMR (DMSO-d6, 400 MHz): δ ppm 8.03 (bs,
1H, NH), 8.02 (d, J = 8.8 Hz, 2H, CHAr), 6.74 (d, J = 8.8 Hz, 2H,
CHAr), 5.28 (q, J = 3.4 Hz, 1H, CH), 2.41–2.23 (m, 2H, CH2CH2CH),
2.18–2.13 (m, 1H, CH2CH2CH), 1.89–1.85 (m, 1H, CH2CH2CH); 13C-
NMR (DMSO-d6, 100 MHz): δ ppm 176.9 (C=O), 153.2 (C), 137.0 (C),
126.5 (3 CHAr), 112.1 (CHAr), 63.6 (CH), 29.2 (CH2), 28.2 (CH2). IR ν
(cm−1): 3349, 3259, 1681, 1594, 1477, 1326, 1254, 1176, 1121.
2.2.32. 5-((4-Nitronaphthalen-1-yl)amino)pyrrolidin-2-one (3p)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the wanted compound as a white solid (2.5 g,
53% yield); m.p. 204–205 °C; 1H-NMR (DMSO-d6, 400 MHz): δ ppm
8.83 (dd, J = 9.2 Hz, J = 1.0 Hz, 1H, NH), 8.48 (d, J = 8.0 Hz, 2H,
CHAr), 8.40 (d, J = 8.0 Hz, 1H, CHAr), 7.90 (d, J = 8.0 Hz, 1H, CHAr),
7.77 (d, J = 8.0 Hz, 1H, CHAr), 7.61 (t, J = 8.0 Hz, 1H, CHAr), 6.77 (d,
J = 9.2 Hz, 1H, NH), 5.52 (t, J = 5.1 Hz, 1H, CH), 2.45–2.35 (m, 2H,
CH2CH2CH), 2.19–2.15 (m, 2H, CH2CH2CH); 13C-NMR (DMSO-d6,
100 MHz): δ ppm 176.7 (C=O), 149.2 (C), 133.5 (C), 130.0 (CHAr),
129.5 (CHAr), 127.0 (C), 125.4 (CHAr), 123.3 (CHAr), 123.0 (CHAr),
121.7 (C), 102.5 (CHAr), 63.8 (CH), 28.8 (CH2), 27.3 (CH2). IR ν
(cm−1): 3402, 3164, 3079, 1692, 1573, 1482, 1253, 1182.
2.2.28. 5-(Methyl(4-nitrophenyl)amino)pyrrolidin-2-one (3 l)
Following the general procedure, the crude product was purified
through flash liquid chromatography being eluted with a gradient of n-
heptane/ethyl acetate, the wanted compound being eluted in 90% ethyl
acetate, as 1.6 g of a yellow solid, in 40% yield; m.p. 152–153 °C; 1H-
NMR (DMSO-d6, 400 MHz): δ ppm 8.22 (s, 1H, NH), 8.08 (d,
J = 9.5 Hz, 2H, CHAr), 6.98 (d, J = 9.5 Hz, 2H, CHAr), 5.83 (q,
J = 3.4 Hz, 1H, CH), 2.82 (s, 3H, CH3), 2.45–2.37 (m, 2H, CH2CH2CH),
2.2.33. 5-(Naphthalen-1-ylamino)pyrrolidin-2-one (3q)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
7

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
(2 × 10 mL), affording the wanted compound as a white solid (2.4 g,
60% yield); m.p. 137–138 °C; 1H-NMR (DMSO-d6, 400 MHz): δ ppm
8.39 (bs, 1H, NH), 8.21 (d, J = 8.0 Hz, 1H, CHAr), 7.77 (d, J = 8.0 Hz,
1H, CHAr), 7.46–7.39 (m, 2H, CHAr), 7.27 (t, J = 7.6 Hz, 1H, CHAr),
7.19 (d, J = 7.6 Hz, 1H, CHAr), 6.65 (d, J = 7.6 Hz, 1H, CHAr), 6.46
(d, J = 8.4 Hz, 1H, NH), 5.31 (d, J = 3.4 Hz, 1H, CH), 2.45–2.32 (m,
2H, CH2CH2CH), 2.11–2.09 (m, 2H, CH2CH2CH); 13C-NMR (DMSO-d6,
100 MHz): δ ppm 176.5 (C=O), 141.9 (C), 133.9 (C), 127.8 (CHAr),
126.5 (CHAr), 125.6 (CHAr), 124.0 (CHAr), 123.2 (CHAr), 121.9
(CHAr), 116.7 (C), 105.0 (CHAr), 64.1 (CH), 29.1 (CH2), 27.7 (CH2). IR
ν (cm−1): 3353, 3048, 1702, 1579, 1530, 1409, 1258.
CDCl3): δ 176.2 (C=O), 157.1 (C), 148.0 (CHAr), 137.4 (CHAr), 114.4
(CHAr), 109.0 (CHAr), 62.9 (CH), 29.0 (CH2), 28.1 (CH2). IR ν (cm−1):
3416, 3211, 1679, 1599, 1525, 1484, 1284, 1266. Anal. Calcd for
C9H11N3O: C, 61.00; H, 6.26; N, 23.71; found: C, 60.86; H, 6.36; N,
23.43%.
2.2.38. 5-((5-Methylthiazol-2-yl)amino)pyrrolidin-2-one (4b)
Following the general procedure, the precipitated product was
washed with ether several times in order to afford the wanted com-
pound, as a white solid (1.85 g, 54% yield); m.p. 166–167 °C; 1H NMR
(DMSO-d6, 400 MHz): δ 7.82 (s, 1H, NH), 7.63 (s, 1H, NH), 6.69 (s, 1H,
CH), 5.29 (s, 1H, CH), 2.50 (bs, 2H, CH2CH2CH), 2.30 (s, 3H, CH3), 2.10
(bs, 1H, CH2CH2CH), 1.88 (bs, 1H, CH2CH2CH); 13C{1H}NMR (DMSO-
d6, 100 MHz): δ 176.5 (C=O), 166.2 (C), 136.0 (CH), 120.8 (C), 65.4
(CH), 29.2 (CH2), 28.1 (CH2). IR ν (cm−1): 3280, 1670, 1450, 1280,
1109.
2.2.34. 5-((4-(((5-Methylisoxazol-3-yl)methyl)sulfonyl)phenyl) amino)
pyrrolidin-2-one (3r)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (30 mL), filtered off and washed with ethanol
(3 × 10 mL), affording the compound as a white solid (3.2 g, 55%
yield); m.p. 231–232 °C; 1H NMR (DMSO-d6, 400 MHz): δ 11.00 (bs,
1H, NH), 8.36 (s, 1H, NH), 7.56 (d, J = 8.0 Hz, 2H, CHAr), 7.12 (d,
J = 8.0 Hz, 1H, CHAr), 6.71 (d, J = 8.0 Hz, 2H, CHAr), 6.11 (s, 1H,
NH), 5.18 (bs, 1H, CH), 2.50–2.34 (m, 2H, CH2CH2CH), 2.28 (s, 3H,
CH3), 2.1–2.12 (m, 2H, CH2CH2CH), 1.84–1.81 (m, 2H, CH2CH2CH);
13C{1H}NMR (DMSO-d6, 100 MHz): δ 176.8 (C), 170.4 (C), 158.3 (C),
151.1 (C), 129.1 (2 CHAr), 126.1 (C), 112.3 (2 CHAr), 95.7 (CHAr),
63.5 (CH), 29.4 (CH2), 28.3 (CH2), 12.5 (CH3). IR ν (cm−1): 3387,
3220, 1681, 1596, 1325, 1157, 1095.
2.2.39. 2,5-Dimethyl-4-((5-oxopyrrolidin-2-yl)amino)-1-phenyl-1H-
pyrazol-3(2H)-one (4c)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 4c as a light-yellow solid (3.8 g, 77%
yield); m.p. 149–150 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.00 (bs, 1H,
NH), 7.43 (t, J = 8.0 Hz, 2H, CHAr), 7.34 (d, J = 8.0 Hz, 2H, CHAr),
7.23 (t, J = 8.0 Hz, 1H, CHAr), 4.90 (bs, 1H, CH), 4.25 (d, J = 8.8 Hz,
1H, NH), 2.84 (s, 3H, CH3), 2.26–2.18 (m, 2H, CH2CH2CH), 2.12 (s, 3H,
CH3), 2.02–1.99 (m, 1H, CH2CH2CH), 1.82–1.80 (m, 1H, CH2CH2CH);
13C{1H}NMR (DMSO-d6, 100 MHz): δ 176.4 (C=O), 163.0 (C=O),
146.2 (C), 135.9 (C), 129.4 (2 CHAr), 126.0 (2 CHAr), 123.1 (CHAr),
117.6 (C), 67.7 (CH), 37.5 (CH3), 29.7 (CH2), 28.3 (CH2), 10.7 (CH3).
2.2.35. 5-((3-(Trifluoromethyl)phenyl)amino)pyrrolidin-2-one (3 s)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the wanted compound as an orange solid (1.4 g,
33% yield); m.p. 149–150 °C. 1H NMR (DMSO-d6, 400 MHz): δ 8.40 (s,
1H, NH), 7.32 (t, J = 8.2 Hz, 1H, CHAr), 6.91 (d, J = 8.2 Hz, 2H,
CHAr), 6.87 (s, 1H, NH), 6.65 (d, J = 8.2 Hz, CHAr), 5.19 (td,
J = 8.0 Hz, J = 3.5 Hz, 1H, CH), 2.41–2.20 (m, 2H, CH2CH2CH),
2.10–2.03 (m, 1H, CH2CH2CH), 1.80–1.75 (m, 1H, CH2CH2CH);
13C{1H}NMR (DMSO-d6, 100 MHz): δ 176.8 (C=O), 147.7 (C), 130
(CHAr), 129.0 (q, JC-F = 31.2 Hz, C), 124.8 (q, JC-F = 272.0 Hz, C),
116.7 (CHAr), 113.0 (d, JC-F = 3.8 Hz, CHAr), 109.3 (d, JC-F = 3.8 Hz,
CHAr), 63.9 (CH), 29.5 (CH2), 28.4 (CH2). IR ν (cm−1): 3314, 1695,
1612, 1451, 1286, 1107, 1068.
2.2.40. 5-((8-Hydroxynaphthalen-2-yl)amino)pyrrolidin-2-one (4d)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the compound as a white solid (1.9 g, 45%
yield); m.p. 122–123 °C; 1H-NMR (DMSO-d6, 400 MHz): δ ppm 8.95 (bs,
1H, NH), 7.84 (d, J = 8.8 Hz, 1H, CHAr), 7.55 (bs, 1H, CHAr),
7.04–6.98 (m, 2H, CHAr), 6.84 (dd, J = 6.9 Hz, J = 1.9 Hz, 1H, CHAr),
6.73 (d, J = 8.8 Hz, 1H, CHAr), 5.63 (bs, 1H, CH), 3.45 (bs, 1H, OH),
2.32–2.40 (m, 2H, CH2CH2CH), 2.17–2.08 (m, 1H, CH2CH2CH),
1.96–1.90 (m, 1H, CH2CH2CH); 13C-NMR (DMSO-d6, 100 MHz): δ ppm
176.3 (C=O), 154.6 (C), 137.7 (C), 136.6 (C), 136.6 (CHAr), 123.5
(CHAr), 122.2 (CHAr), 116.5 (C), 112.8 (CHAr), 111.8 (CHAr), 61.8
(CH), 29.1 (CH2), 27.4 (CH2). IR ν (cm−1): 3364, 3161, 1694, 1612,
1529, 1266, 1241.
2.2.36. 5-((4-Acetylphenyl)amino)pyrrolidin-2-one (3 t)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 3 t as a white solid (2.6 g, 70%
yield); m.p. 171–172 °C; 1H-NMR (DMSO-d6, 400 MHz): δ ppm 7.74 (d,
J = 8.4 Hz, 2H, ArH), 6.68 (d, J = 8.4 Hz, 2H, ArH), 5.22 (td,
J = 6.8 Hz, J = 3.2 Hz, 1H, CH), 2.36–2.29 (m, 2H, CH2CH2CH), 2.35
(s, 3H, CH3), 2.16–2.11 (m, 1H, CH2CH2CH), 1.87–1.83 (m, 1H,
CH2CH2CH); 13C-NMR (DMSO-d6, 100 MHz): δ ppm 195.7 (C=O),
176.8 (C=O), 151.4 (C), 130.8 (2CHAr), 126.2 (C), 112.1 (2CHAr),
63.6 (CH), 29.4 (CH2), 28.3 (CH2), 26.4 (CH3). IR ν (cm−1): 3280,
1686, 1576, 1424, 1240, 1162. Anal. Calcd for C12H14N2O2: C, 66.04;
H, 6.47; N, 12.84, found: C, 66.35; H, 6.31; N, 12.44%.
2.2.41. 1-(5-Oxopyrrolidin-2-yl)indoline-2,3-dione (4e)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 4e as an orange solid (2.6 g, 65%
yield); m.p. 187–188 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.14 (s, 1H,
NH), 7.68 (td, J = 7.64 Hz, J = 1.30 Hz, 1H, CHAr), 7.61 (dd,
J = 7.64 Hz, J = 1.30 Hz, 1H, CHAr), 7.17 (t, J = 7.6 Hz, 1H, CHAr),
7.15 (d, J = 7.6 Hz, 1H, CHAr), 5.91 (dd, J = 9.0 Hz, J = 3.2 Hz, 1H,
CH), 2.65–2.53 (m, 2H, CH2CH2CH), 2.34–2.20 (m, 2H, CH2CH2CH);
13C{1H}NMR (DMSO-d6, 100 MHz): δ 183.5 (C=O), 177.2 (C=O),
158.23 (C=O), 149.59 (C), 138.62 (CHAr), 125.27 (CHAr), 123.78
(CHAr), 118.23 (C), 111.96 (CHAr), 62.6 (CH), 29.7 (CH2), 24.1 (CH2).
IR ν (cm−1): 3216, 1742, 1679, 1606, 1468, 1254. Anal. Calcd for
2.2.37. 5-(Pyridin-2-ylamino)pyrrolidin-2-one (4a)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording compound 4a as a white solid (4.6 g, 60%
yield); m.p. 158–159 °C; 1H-NMR (400 MHz, CDCl3): δ 8.10 (d,
J = 5.8 Hz, 1H, CHAr), 7.45 (td, J = 8.1 Hz, J = 1.6 Hz, 1H, CHAr),
6.68 (t, J = 8.1 Hz, 1H, CHAr), 6.59 (bs, 1H, NH), 6.45 (d, J = 8.1 Hz,
1H, CHAr), 5.59 (td, J = 7.4 Hz, J = 4.9 Hz, 1H, CH), 4.74 (d,
J = 7.0 Hz, 1H, NH), 2.62–2.41 (m, 2H, CH2CH2CH), 2.40–2.35 (m,
1H, CH2CH2CH), 1.90–1.61 (m, 1H, CH2CH2CH); 13C-NMR (100 MHz,
C
12H10N2O3: C, 62.60; H, 4.38; N, 12.17; found: C, 62.26; H, 4.17; N,
11.82%.
2.2.42. 3-(5-Oxopyrrolidin-2-yl)thiazolidine-2,4-dione (4f)
Following the general procedure, the crude product was pre-
cipitated in diethyl ether (20 mL), filtered off and washed with ethanol
(2 × 10 mL), affording the wanted compound as a white solid (2.3 g,
8

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
67% yield); m.p. 186–187 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.05 (s,
1H, NH), 5.68 (d, J = 8.9 Hz, 1H, CH), 2.60–2.39 (m, 4H, CH2CH2CH,
O=CCH2CH2C=O), 2.20–2.00 (m, 2H, CH2CH2CH); 13C{1H}NMR
(DMSO-d6, 100 MHz): δ 177.7 (C=O), 172.0 (C=O), 171.6 (C=O),
63.2 (CH), 33.5 (CH2), 29.4 (CH2), 24.1 (CH2). IR ν (cm−1): 3037,
2943, 1747, 1662, 1277, 1149. Anal. Calcd for C7H8N2O3S, 0.5H2O: C,
40.18; H, 4.34; N, 13.39; S, 15.33; found: C, 40.57; H, 3.93; N, 13.52; S,
15.28%
3. Results and discussion
3.1. Synthetic chemistry
This study needs the introduction of a heteroatom linked to an alkyl
or aryl group to the position 5 of pyrrolidin-2-one. That can be realized
in the case of few amines by reacting them directly with the lactam in
oxidative and metal catalyzed conditions [31]. A more general reaction
started from pterolactam A relying on the use of a triflic acid catalyzed
intermolecular α-amination of pterolactam via N-acyliminium species
to produce 5-arylamino pyrrolidinones [32]. However the scope of this
reaction is limited. In fact, this acidic protocol gave good yields with
anilines, but failed when using aliphatic amines or benzyl alcohol de-
rivatives for instance. We recently described a new procedure for the
reaction of pterolactam with nucleophiles in non-acidic conditions
[25]. Using this method, the starting nucleophile and a catalytic
amount of cesium fluoride were reacted under a slight vacuum, with
lactam A at 80 °C. With this very efficient method, all target compounds
were obtained in moderate to good yields and gave analytical and
spectroscopic data in full accordance with their structures. Four series
of N,O-, N,S-acetals and N,N′-aminals derivated from natural pter-
olactam A, structurally similar to some plant metabolites, were ob-
tained and tested against a panel of fungal strains and yeasts.
2.3. Strains and culture conditions
The antifungal activities of the synthesized compounds were tested
in vitro against a total of twelve species. Eight phytopathogenic fungal
strains and four yeast strains were selected from the collection of our
mycology laboratory. The strains Aspergillus oryzae (MUCL19009),
Alternaria alternata (MUCL53651), Paecilomyces variotii (MUCL 39890),
Penicillium ochrochloron (MUCL 38775), Sclerotinia sclerotiorum (MUCL
011553), Fusarium solani (MUCL 035016), Cladosporium cladosporioides
(Laboratory's isolate), Botrytis cinerea (Laboratory's isolate), and yeasts
Geotrichum candidum (Laboratory's isolate), Candida boratory's isolate),
Candida
pseudotropicalis
(MUCL46196),
Candida
tropicalis
(MUCL29893), packed in tubes on PDA medium, were further plated on
Petri dishes containing potato dextrose agar, and incubated at 22 °C for
5 to 7 days so that pure young cultures could be obtained, with periodic
subculturing.
3.2. Antifungal activity and structure-activity relationships (SAR)
2.4. Culture procedures
The in vitro antifungal activities of these original compounds were
evaluated using the micro-dilution method, by using hymexazol - a
systemic soil and seed broad spectrum fungicide -, fluconazole and
ketoconazole as the positive controls. A panel of twelve fungi (Fusarium
solani, Paecilomyces variotii, Penicillium ochrochloron, Aspergillus oryzae,
Alternaria alternata, Cladosporium cladosporioides, Geotrichum candidum,
Sclerotinia sclerotiorum and Botrytis cinerea) known for causing allergies,
asthma, mycosis or plant pathologies, and also non albicans candida
yeasts species (Candida pseudotropicalis, Candida tropicalis and Candida
krusei) which have demonstrated reduced susceptibility to commonly
used antifungal drugs was selected. 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 (EC50) values of products
with high inhibition rate (> 75%) were determined (Table 2).
Data presented in Table 1 indicate that the Sclerotiniaceae family (S.
sclerotiorum and B. cinerea) and C. cladosporiodoides are not sensitive to
the new Mannich base of amides inspired by pterolactam. However, in
term of antifungal activity, a third of the newly synthesized compounds
displayed antifungal activities against at least one strain (Tables 1–2).
The most sensitive strains to the tested compounds were P. ochrochloron
and F. solani, with five and four compounds displaying EC50 in the same
range as for the control (EC50: 0.077–0.236 vs 0.628 μmol/mL and
0.050–0.357 vs 0.157 μmol/mL respectively) (Table 2, lines PO and FS).
This result is of particular interest for F. solani considering the described
problems of drug resistance in this species for both human health and
agricultural purposes [33]. It is also notable than some compounds can
be considered as broad-spectrum antifungal agents (ie compounds 1i,
3o and 3p) whereas others targeted selectively one pathogenic strain (ie
compounds 1f, 3 l, 4c) (Table 2, entries 2–4, 14–18, 19–22, 1, 12, 25,
respectively).
The mineral salts medium consisted of (g/L): KCl, 0.5; NaH2PO4,
1.544; Na2HPO4, 0.008; MgSO4, 0.244; NH4NO3, 1; and trace-element
solution consisting of (mg/L): ZnSO4.7H2O, 1; MnCl2.4H2O, 0.1;
FeSO4.7H2O, 1; CuSO4.5H2O, 0.5; CaCl2.2H2O, 0.1; MoO3, 0.2. The
carbon source was glucose at 10 g/L. Each fungal isolate was singly
grown in Erlenmeyer flasks (500 mL) for one week at 22 °C at 140 rpm
under a 12 h/12 h photoperiod in a Binder incubator including an or-
bital shaker. Fungi broth microdilutions were performed in 96-well
microtiter plates. For the assay, compound test wells (CTWs) were
prepared with stock solutions of each compound in DMSO. The com-
pounds possessing good activity (inhibitory rate above 75% at 100 μg/
mL) were further evaluated using different concentrations by diluting
the above stock solution in the range 1 to 100 μg/mL. An inoculum
suspension (100 μL) was added to each well (final volume in the
well = 200 μL). A growth control well (GCW) (containing medium,
inoculum and the same amount of DMSO used in a CTW) was included
for each fungus tested. Microtiter trays were incubated in a moist
chamber at 22 °C for 5 to 7 days for all yeasts. DMSO served as the
negative control, having no observed influence on the tested panel. The
cultures were incubated for 7 days, and turbidity was measured through
the optical density using a microplate reader (Multiskan Go, Thermo),
at 600 nm. Tests were performed in triplicates.
2.5. Cell culture and cytotoxic assay
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/strepto-
mycin, non-essential amino acid solution (1/100) and 5% (v/v) heat-
inactivated fetal 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 pro-
liferation and cytotoxicity) was estimated by the colorimetric MTS (3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium) assay.
In the Series a of ethers and thioethers it is interesting to note that
only a third of the compounds exhibited some activity against at least
one strain at 100 μg/mL (Table 1, entries 1–10). Sessiline 1 g [26] as
well as the other benzyl ethers 1b-e substituted by oxygenated groups
demonstrated no activity. Compound 1f, bearing a benzodioxole ring
appeared three times more potent than the control against P. ochro-
chloron (EC50: 0.136 vs 0.628 μmol/mL) (Table 2, entry 1). This com-
pound is structurally similar to sesamol, known for its antimycotic
properties [34]. Concerning the benzhydryl ether derivatives 1 h and 1i
9

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
Table 2
EC50 (μmol/mL) of selected compounds against the sensitive fungi and yeasts.d
Entry Compound Structure
M (g/mol) Strain Toxic regression
R2
EC50 (μmol/mL) Reference EC50 (μmol/mL) Spectrume
(y = ax+b)
a
1
1f
1i
235.24
336.21
PO
y = 291.48× + 10.102
0.9840 0.136
0.628
1
3
a
c
b
2
3
4
PV
GC
CK
y = 1195× + 32.26
y = 623.33× + 46.927
y = 225.74× + 41.12
0.9708 0.014
0.9216 0.005
0.9525 0.039
0.577
0.003
0.045
a
c
5
6
1j
196.03
FS
y = 412.51×-8.1445
y = 683.24×-1.5085
0.9772 0.139
0.9772 0.076
0.157
0.019
2
CP
a
7
2d
2e
263.31
204.27
FS
11% inhibition rate at 50 μg/mL
–
> 0.3
0.157
1
1
a
8
FS
No inhibition rate at 50 μg/mL
–
> 0.3
0.157
a
9
2 g
224.69
FS
20% inhibition rate at 50 μg/mL
–
> 0.3
0.157
1
a
a
a
10
11
12
3c
192.21
221.21
235.24
FS
FS
FS
y = 307.54×-60
1
–
0.357
> 0.3
0.157
0.157
0.157
1
1
1
3 k
3 l
15% inhibition rate at 50 μg/mL
y = 134.53× + 28.047
0.9901 0.164
a
13
3 m
3o
237.21
251.24
FS
No inhibition rate at 50 μg/mL
–
> 0.3
0.157
1
5
a
a
a
c
c
a
a
a
b
a
a
14
15
16
17
18
19
20
21
22
23
24
PV
PO
AA
CP
CT
AO
PO
FS
y = 3140.5× + 37.5
y = 301.49× + 15.75
y = 2263.2×-4.377
y = 250.96×-0.4469
y = 319.85×-30.718
y = 219.87× + 6.9737
y = 254.99× + 30.4
y = 907.81× + 3.8812
y = 334.05× + 32.880
y = 415.87–48
1
0.004
0.577
0.628
0.387
0.019
0.030
0.461
0.628
0.157
0.045
0.628
0.387
0.9985 0.114
0.9693 0.024
0.9989 0.201
0.9489 0.252
0.9990 0.195
0.8982 0.077
0.9466 0.050
0.8937 0.051
3p
271.27
4
CK
PO
AA
3r
4c
335.38
286.33
1
0.236
2
1
y = 2202× + 37.984
0.9455 0.005
a
25
FS
y = 248.3 × 24.055
0.9419 0.104
0.157
SS - Sclerotinia sclerotiorum; BC - Botrytis cinerea; AO - Aspergillus oryzae; PV - Paecilomyces variotii; PO - Penicillium ochrochloron; CC - Cladosporium cladosporioides; AA -
Alternaria alternata; FS - Fusarium solani; GC - Geotrichum candidum; CK - Candida krusei; CP - Candida pseudotropicalis; CT - Candida tropicalis.
a
b
c
d
Hymexazol, used as positive control for bioassay.
Fluconazole, used as positive control for bioassay.
Ketoconazole, used as positive control for bioassay.
Sequential dilutions from 100 μg/mL to 1 μg/mL were performed for bioassays.
e
Spectrum refers to the number of strains killed by the compound.
it is notable that the dichloro compound 1i is highly active whereas 1 h
is not (Table 1, entries 8 and 9). The dichlorobenzhydryl product 1i
proved to be as active as the control against G. candidum and C. krusei
but above all fifty time more potent as the control against P. variotii
(EC50: 0.014 vs 0.577 μmol/mL, Table 2, entry 2). Turning to the
thiophenyl ether, introduction of the sulfur linkage furnished 1j with
the same activity as the control against F. solani but four time less
against C. pseudotropicalis (EC50 0.076 μmol/mL) (Table 2, entry 3).
Concerning Series b of aliphatic amino-5-pyrrolidone derivatives,
even if some inhibition rates were observed for compounds 2d, 2e and
2 g at 100 μg/mL against F. solani, their EC50 were above 0.3 μmol/mL
and, consequently, they were considered as inactive (Table 1, entries
14, 15 and 17 and Table 2, entries 7–9). Almost the same considerations
can be provided for the heterocycles of the Series d, for which only the
Mannich base 4c combining a pyrrolidin-2-one and phenyl dimethyl
pyrazole moieties proved to be slightly more active as the control
against F. solani (EC50: 0.104 vis 0.157 μmol/mL).
The Series c of aromatic amines Mannich bases furnished the more
promising compounds (Table 2, entries 10–24). Indeed, while the
product 3a issued from aniline proved to be inactive, addition of an OH
group in para position led to 3c, two times less potent against F. solani
than hymexazol (EC50: 0.357 vs 0.157 μmol/mL). Exchanging the OH
for the nitro group of 3 k did not help increasing the activity (Table 2,
entry 11).
The N-methylation of the linker, leading to compound 3 l slightly
increased the activity (EC50: 0.164 μmol/mL) compared to 3 k (Table 2,
entry 12). Now, replacing the nitro-phenyl group of 3 k by the bulkier
nitro-naphthalene yielded 3p. Not only this compound is three time
more potent as the control against F. solani (EC50: 0.050 μmol/mL), but
it is also equal or eight time more active as the control against A. oryzae,
P. ochrochloron and C. krusei (EC50: 0.195 vs 0.461 μmol/mL, 0.077 vis
0.628 μmol/mL and 0.051 vis 0.045 μmol/mL respectively) (Table 2,
entries 19–22). This increase of activity comes from the nitro group
because the naphtyl analog 3q displayed no biological properties
10

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
(Table 1, entry 35). Noteworthy also, the para-sulfamide withdrawing
group led to 3r which displayed two and ten times more activity against
P. ochrochloron and A. alternata (EC50: 0.236 and 0.005 μmol/mL re-
spectively). The modification of the substitution on the aromatic ring is
not well tolerated. Exchanging the NO2 group for a methyl or a halogen
substituent abolished the activity (Table 1, entries 2, 25–26).
The same observation was done when varying the position of the
NO2 group from para to ortho or meta (Table 1, entries 28–30). More-
over, keeping the para-nitro group and adding a second substituent on
the aromatic ring – cyano or hydroxy - led to no activity (Table 1, en-
tries 31–32). However, Mannich base 3o, formed from pyrrolidin-2-one
and 3-nitro-5-methoxy aniline become inactive against F. solani, and ten
times less potent as the control against C. pseudotropicalis and C. tropi-
calis while becoming five and fifty times more active as the control
against P. ochrochloron and A. alternata and even provide a very im-
pressive 0.004 μmol/mL EC50 value against P. variotii (control:
0.58 μmol/mL) (Table 2, entry 11). Thus, compound 3o prove to be a
very interesting platform molecule because his broad antimicrobial
spectrum (five sensitive strains among twelve tested).
Fig. 2. HEK293 cell line (TPP culture plates) cell viability of selected molecules
tested at 100 μM concentration.
spectrum antimicrobial discovered agent exhibited no cytotoxicity.
However, all compounds showed no to a slight toxicity in viable kidney
cells, the viability of cells dropping down around 40% for the most toxic
compounds at a concentration of 100 μM. On the other side, fifteen
compounds (1b, 1d, 1j, 2a, 2f, 2a, 3c, 3f, 3 k, 3o, 4b, 4c, 4d, 4e, 4f)
were selected by the National Cancer Institute (NCI) for screening on a
large panel of cancer cells, and they exhibited slight to no cytotoxicity
(see data in ESI).
3.3. Molecular physicochemical properties of the selected antimicrobial
agents (Table 3)
To further evaluate their potential for candidate lead fungicide,
some of the molecular physicochemical properties [octanol/ water
partition coefficient (LogP), hydrophilic–lipophilic balance (HLB), to-
pology 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) [35]. Except 1i, all these
compounds meet the Lipinski “Rule of five” criteria [36] with Log
P < 1.8, NDS < 4, NAS < 9 and MW < 350, and the Briggs
“Ground rules of three” suggesting that for a fungicidal to be available,
it should have a Log P < 3 [37]. It can also be observed that most of
the active compounds are lipophilic. However, no obvious correlation
can be found between the antifungal activity over a particular fungal
species and only one of these parameters.
4. Conclusions
The inhibition activity of 43 hemisynthetic compounds inspired
from natural pterolactam and its metabolites were evaluated against a
large panel of fungal strains and yeasts, some of them being particularly
sensitive to this class of compounds. According to the structure-activity
relationships, N,N′-aminals from pterolactam were identified as more
potent antimicrobial agents than N,O- or N,S-acetals. Moreover, aro-
matics bearing a nitro-group were of particular interest, especially to
fight against F. solani. This screening underlined that compound 3o,
combining a pyrrolidin-2-one and a 3-methoxy-5-nitroaniline moieties,
is a broad-spectrum antifungal agent with adequate “fungicide-like”
properties and no visible cytotoxicity.
3.4. Toxicity evaluation
In order to check the mammalian cell toxicity of the tested com-
pounds presented herein, the most active products were screened
against human embryonic kidney cells (HEK293) at a very high con-
centration of 100 μM (Fig. 2) [38]. Compound 3o, the broader-
Table 3
Predicted molecular properties of the new compounds.
Compound
Log Pa
HLBb
TPSAc
NDSd
NASe
MWf
Sensible strains
Spectrum
1f
0.82
4.19
1.56
1.42
1.17
1.48
0.49
0.73
1.37
0.43
0.57
1.72
0.62
0.11
11.4
56.79
38.33
29.10
35.58
41.11
41.13
61.36
86.95
78.16
107.18
96.18
86.95
100.44
64.68
1
1
1
1
2
2
3
2
1
3
2
2
3
2
4
2
1
3
2
2
3
4
4
5
5
4
4
4
235.24
336.21
193.26
263.32
204.27
224.69
192.22
221.21
235.24
237.21
251.24
271.28
335.38
286.33
PO
1
3
2
1
1
1
1
1
1
1
5
4
2
1
1i
5.42
PV – GC - CK
1j
8.39
FS - CP
2d
2e
2 g
3c
3 k
3 l
3 m
3o
3p
3r
14.38
11.87
12.56
9.06
FS
FS
FS
FS
10.11
10.29
11.77
11.94
8.03
FS
FS
FS
PV – PO – AA – CT - CP
AO – PO – FS – CK
PO – AA
8.98
4c
19.44
FS
a
Log P = octanol/water partition coefficient.
b
c
d
e
f
Hydrophilic–lipophilic balance (HLB) is the balance of the size and strength of the hydrophilic and lipophilic moieties of a compound.
TPSA = topology polar surface area.
NDS = number of hydrogen bond donor sites.
NAS = number of hydrogen bond acceptors sites.
MW = molecular weight (g/mol).
11

A.-E. Dascalu, et al.
Fitoterapia143(2020)104581
Declaration of Competing Interest
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