Natural α-methylenelactam analogues: Design, synthesis and evaluation of α-alkenyl-γ and δ-lactams as potential antifungal agents against Colletotrichum orbiculare

Article abstract of DOI:10.1016/j.ejmech.2017.02.050

In our continued efforts to improve the potential utility of the α-methylene-γ-lactone scaffold, 62 new and 59 known natural α-methylenelactam analogues including α-methylene-γ-lactams, α-arylidene-γ and δ-lactams, and 3-arylideneindolin-2-ones were synthesized as the bioisosteric analogues of the α-methylenelactone scaffold. The results of antifungal and cytotoxic activity indicated that among these derivatives compound (E)-1-(2, 6-dichlorobenzyl)-3-(2-fluorobenzylidene) pyrrolidin-2-one (Py51) possessed good selectivity with the highest antifungal activity against Colletotrichum orbiculare with IC₅₀?=?10.4?μM but less cytotoxic activity with IC₅₀?=?141.2?μM (against HepG2 cell line) and 161.2?μM (against human hepatic L02?cell line). Ultrastructural change studies performed by transmission electron microscope showed that Py51 could cause important cell morphological changes in C.?orbiculare, such as plasma membrane detached from cell wall, cell wall thickening, mitochondria disruption, a dramatic increase in vacuolation, and eventually a complete loss in the integrity of organelles. Significantly, mitochondria appeared one of the primary targets, as confirmed by their remarkably aberrant morphological changes. Analysis of structure–activity relationships revealed that incorporation of the aryl group into the α-exo-methylene and the N-benzyl substitution increased the activity. Meanwhile, the α-arylidene-γ-lactams have superiority in selectivity over the 3-arylideneindolin-2-ones. Based on the results, the N-benzyl substituted α-(2-fluorophenyl)-γ-lactam was identified as the most promising natural-based scaffold for further discovering and developing improved crop-protection agents.

Full text of DOI:10.1016/j.ejmech.2017.02.050

Accepted Manuscript
Natural α-methylenelactam analogues: Design, synthesis and evaluation of α-alkenyl-
γ and δ-lactams as potential antifungal agents against Colletotrichum orbiculare
Wang Delong, Wang Lanying, Wu Yongling, Song Shuang, Feng Juntao, Zhang Xing
PII:
S0223-5234(17)30123-X
10.1016/j.ejmech.2017.02.050
EJMECH 9246
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 November 2016
Revised Date: 18 February 2017
Accepted Date: 20 February 2017
Please cite this article as: W. Delong, W. Lanying, W. Yongling, S. Shuang, F. Juntao, Z. Xing, Natural
α-methylenelactam analogues: Design, synthesis and evaluation of α-alkenyl-γ and δ-lactams as
potential antifungal agents against Colletotrichum orbiculare, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.02.050.
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Graphical Abstract

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Natural α-methylenelactam analogues: Design, synthesis and evaluation of α-alkenyl-γ and
δ-lactams as potential antifungal agents against Colletotrichum orbiculare
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a, b
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Wang Delong , Wang Lanying , Wu Yongling , Song Shuang , Feng Juntao *, Zhang Xing
Authors’ address:
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Research & Development Center of Biorational Pesticide, Key Laboratory of Plant Protection
Resources and Pest Management of Ministry of Education, Northwest A&F University, Yangling
712100, China.
b
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College of Environment and Plant Protection, Hainan University, Haikou, Hainan, 570228, China
*Correspondence to this author (telephone +86-29-87092122; fax +86-29-87092122; E-Mail:
fengjt@nwafu.edu.cn).
Abstract
In our continued efforts to improve the potential utility of the α-methylene-γ-lactone scaffold, 62
new and 59 known natural α-methylenelactam analogues including α-methylene-γ-lactams,
α-arylidene-γ and δ-lactams, and 3-arylideneindolin-2-ones were synthesized as the bioisosteric
analogues of the α-methylenelactone scaffold. The results of antifungal and cytotoxic activity
indicated
that
among
these
derivatives
compound
(E)-1-(2,
6-dichlorobenzyl)-3-(2-fluorobenzylidene) pyrrolidin-2-one (Py51) possessed good selectivity with
the highest antifungal activity against Colletotrichum orbiculare with IC50 = 10.4 µM but less
cytotoxic activity with IC = 141.2 µM (against HepG2 cell line) and 161.2 µM (against human
50
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hepatic L02 cell line). Ultrastructural change studies performed by transmission electron
microscope showed that Py51 could cause important cell morphological changes in C. orbiculare,
such as plasma membrane detached from cell wall, cell wall thickening, mitochondria disruption, a
dramatic increase in vacuolation, and eventually a complete loss in the integrity of organelles.
Significantly, mitochondria appeared one of the primary targets, as confirmed by their remarkably
aberrant morphological changes. Analysis of structure–activity relationships revealed that
incorporation of the aryl group into the α-exo-methylene and the N-benzyl substitution increased the
activity. Meanwhile, the α-arylidene-γ-lactams have superiority in selectivity over the
3-arylideneindolin-2-ones.
Based
on
the
results,
the
N-benzyl
substituted
α-(2-fluorophenyl)-γ-lactam was identified as the most promising natural-based scaffold for further
discovering and developing improved crop-protection agents.
Keywords: α-Methylenelactam; Antifungal activity; Ultrastructural changes studies; Structure–
activity relationships
1. Introduction
Emerging infectious diseases caused by fungi are increasingly recognized as a worldwide persistent
threat to human, animal and plant health [1-4]. As a consequence of the infection, these diseases
have recently caused the biodiversity loss in some regions, resulting in the severe species decline
and measurable ecosystem-level changes in both animals and plants, and, more importantly, are
jeopardizing the global food security [2, 3]. Moreover, these threats are exaggerated in the
landscape by fungal adaption to new ecosystem, generated by trade and transportation, and by
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climate changes [2]. To ensure the global food security, since 1960s the agrochemical industry
provided agriculture with a vast array of synthetic chemicals for crop protection, including
fungicides. According to their fungicidal effects on specific stages in the life cycle of the fungus,
commercial fungicides could be classified into protective, curative, eradicative, and antisporulation
types (Fig. 1) [5]. Over past decades, due to repeated use of these different types of fungicides with
identical or similar mode of action, there has been a rapid increase in fungicide resistance, which is
tightly associated with the failure of fungal diseases control in crops [6]. Meanwhile, non-target and
environmental hazard of fungicidal chemicals has stimulated great public concern [7-9]. In addition,
more stringent registration for agrochemicals has been implemented [10]. On these grounds, there is
a continuing need for the development of new, effective and safe agrochemicals as the alternatives
for the fungal diseases control in crops.
Natural products are the masterpieces of nature’s chemical laboratories and many of them or
their analogues shared the features of bio- and environmental compatibilities, easy biodegradation,
structural diversities and different mode of actions than existing pesticides [10-13]. That is the
reason why natural product has long been used as pesticides and has broadly served as a source of
inspiration for a great many commercial synthetic pesticides that are in the market today [10-18].
Therefore, the discovery of new antifungal compounds or scaffolds from natural products has been
intensively focused recently as an important alternative for research and development of new
fungicides.
In our original works, carabrone and its alcohol analog carabrol (Fig. 2), two known
sesquiterpene lactones isolated from the fruits of Carpesium macrocephalum, were found to possess
potent antifungal activity against Colletotrichum orbiculare in a protective manner [19]. From the
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perspective of infection process of fungi and disease cycle, the protective antifungal agents,
blocking early penetrative infection process of spores, play a superiorly preventive role in the
subsequent disease development, and thus were liable to minimize the diseases control cost and
crop losses. For this reason, the protective antifungal agents are very helpful in the crop disease
control. Continuously, study of the structure-activity relationships of carabrone revealed that the
bioactive scaffold α-exo-methylene-γ-lactone plays a crucial role in its activity profile [20, 21].
Interestingly enough, the small, less functionalized analogues of naturally occurring
α-methylene-γ-lactone are represented by tulipalin A and B (Fig. 2), which efficaciously replicate
the effective antifungal ability of the complex natural products [22-24]. Noticeably, further
exploitation of carabrone suffers from fussy total procedures. Accordingly, we concisely
synthesized a collection of α-methylene-γ-lactone derivatives by virtue of pharmacophore strategy
[25, 26]. Though some of these derivatives are potent enough as the lead compounds for further
optimization, there has some barriers hindering our effort to further research. Specifically, their
cytotoxicity could not be negligible and they are not stable enough when applied in fields, which
were plausibly attributed to the high electrophilicity of α-methylene-γ-lactone moiety, a Michael
acceptors [27-29]. Thus this has prompted us to continue to seek new analogous template with
improved efficacy and stability, and low toxicity.
α-Methylene-γ-lactam motif, a bioisostere of α-methylene-γ-lactone by the isosteric
replacement of the oxygen with a nitrogen atom, occurs much less common in many natural
products as well as the α-methylene-δ-lactam [30, 31]. Examples of occurring compounds of this
substructure include pukeleimid E [32], anatin and isoanatin [33] (Fig. 3). Natural or synthetic
compounds containing this moiety show important biological activities, such as cytotoxic,
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antitumor and anti-inflammatory activities [30, 31]. Nevertheless, it has been demonstrated that the
α-methylene-γ-lactam scaffold in place of the γ-lactone moiety could help to mitigate their
promiscuous biological toxicity [34]. Meanwhile, arylation of the α-methylene-γ-lactone
substructure rendered α-benzylidene-γ-lactones, and a preliminary biological evaluation indicated
that appropriate substituents on the α-benzylidene-γ-lactone functional group could reduce their
toxicity [35, 36]. On the other hand, the α-benzylidene-γ-lactam could be envisaged as the product
of chain-ring transformation of cinnamamide derivatives, which received considerable attention as a
result of their proven antifungal activity [37, 38] (Fig. 3).
In this context, we firstly synthesized the α-methylene and benzylidene-γ-lactam by the
isosteric replacement of the oxygen with a nitrogen atom, as it leads to a less reactive and hopefully
more stable and selective system. Additionally, supplementary nitrogen atom allows for a variety of
modulation by substituted with different lipophilic chain or aromatic rings for instance. To estimate
the effect of ring conformation and constraints on the activity, a series of natural α-methylenelactam
analogues α-benzylidene-δ-lactams and 3-benzylideneindolin-2-ones were synthesized and their
antifungal activity was tested. Structure-activity relationships were investigated parallelly to
provide a helpful insight into further lead optimization. The cytotoxicity and selectivity were
sufficiently considered in the optimization. To investigate the ultrastructural changes of fungus
induced by the potent analogue Py51 and lay a preliminary foundation for the further antifungal
mechanism studies on these compounds, transmission electron microscope (TEM) analyses were
performed.
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2. Results and discussion
2.1 Chemistry.
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Depending on the design strategy for the targeted scaffolds (Fig. 3), a variety of different
compounds was prepared including α-methylene-γ-lactams, α-arylidene-γ and δ-lactams, and
3-arylideneindolin-2-ones, as outlined in Scheme 1-6. Previously, we reported that the natural
α-methylene-γ-butyrolactone (tulipalin A) together with its analogues exhibited moderate to good
inhibitory activity on the germination of spores of C. orbiculare [25, 26]. On the basis of these
results, we first investigated the influence of bioisosteric replacement of oxygen atom with NH on
the inhibitory efficacy. Thus, α-methylene-γ-lactam was prepared using the known procedure
(Scheme 1) [39, 40]. A two-step sequence encompassing the protection of commercially available
2-pyrrolidone with di-tert-butyl dicarbonate (Boc O) and the reaction of N-Boc-2-pyrrolidone with
2
diethyl oxalate in the presence of sodium hydride (NaH) provided an enolate intermediate.
Subsequently, the enolate intermediate was reacted with paraformaldehyde, followed by elimination
to give the desired 3-methylene functionality. Finally, removal of the N-Boc protection afforded
α-methylene-γ-lactam 1.
Next, we designed a patterned structural study of α-arylidene-γ and δ-lactams to obtain
structure-activity information and to investigate the electrostatic, steric, hydrogen bonding
requirements, and ring size for inhibitory activity. The preparation of these compounds by direct
aldol type condensation of 2-pyrrolidone with aromatic aldehydes, however, seemed less efficient in
comparison with its bioisostere, tulipalin A. Generally, there are two significant reasons for the
impracticable implementation [41]. First, the α-hydrogens are much less acidic than those of
γ-butyrolactone, which readily undergoes base-catalyzed aldol condensation with aromatic
aldehydes. Second, there occur some crossed side reactions, including the Claisen condensation
between the 2-pyrrolidones and addition of the lactam N-H moiety across the aldehyde carbonyl
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group. Nevertheless, it has been reported that a direct aldol condensation of 2-pyrrolidone with
aromatic aldehydes could be accomplished if the lactam nitrogen function were protected with a
sufficient electron withdrawing group, e.g. an acetyl or trifluoroacetyl group [41-43], which would
enhance the acidity of the α-hydrogens and simultaneously protect the N-H moiety from reacting.
Trifluoroacetyl is preferably selected for protection due to its strong electron withdrawing feature
-
and easy detrifluoroacetylation in the presence of OH generated in situ under mild reaction
conditions. Consequently, with the stereochemistry of the exocyclic double bond being exclusively
E [41-43], this method is concise and stereospecific. With regard to the tolerance of substrates, it
was found that N-trifluoroacetyl-2-pyrrolidone reacted smoothly with aromatic aldehydes
possessing electron-releasing or withdrawing substituents in a base-catalyzed aldol-type
condensation when the strong base potassium tert-butoxide (KO Bu) was employed to give the
t
α-arylidene-γ-lactams (Scheme 2). Particularly, the 3-chlorobenzaldehyde gave α,
β-di(3-chlorobenzylidene)-γ-lactam which was identified by 2D NMR and mass spectrometry
(Supporting Information), rather than the α-monosubstituted product. The hydroxyl compound 22
were synthesized through silyl protection of hydroxyl benzaldehyde using tert-butyldimethylsilyl
chloride (TBSCl) and following deprotection of this ether group using tetrabutylammonium fluoride
(TBAF) [44] (Scheme 2). The α-arylidene-δ-lactams were obtained in a similar way, while
3-chlorobenzaldehyde
afforded
the
only
α-monosubstituted
product
α-(3-chlorobenzylidene)-δ-lactam (Scheme 3).
Subsequently, to evaluate the essentiality of the benzo substituent on the β, γ-positions of
γ-lactam ring, we synthesized a collection of 3-benzylideneindolin-2-ones Ox1-28 with different
substituents on the benzylidene ring by a Knoevenagel reaction [45]. The condensation reaction was
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carried out in ethanol with piperidine as base catalyst by reflux. Meanwhile, a set of analogues
Ox29-32 with different substituents on the indoline ring were synthesized to assess the impact of
these substituents on efficacy of the efficiency 3-benzylideneindolin-2-one scaffold [46]. As shown
in Scheme 4, the corresponding aniline was reacted with chloral hydrate (Cl CCH(OH) ) and
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hydroxylamine hydrochloride (NH OHóHCl) in aqueous sodium sulfate (Na SO ) to form
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isonitrosoacetanilide followed by a Sandmeyer reaction with concentrated sulfuric acid to furnish
the desired isatin in good yield. The reduction of corresponding isatin proceeded efficiently and
conveniently with the low-valent Ti(0) reagent instead of the hydrazine hydrate involved in Wolff–
Kishner reaction, giving to the oxidole analogues [47]. Finally, the 3-benzylideneindolin-2-ones
with different substituents on the indoline ring were similarly synthesized by the Knoevenagel
reaction. The 3-substituted indolin-2-ones may exist either the Z or E isomer depending on the
characteristics of the substituents at C-3 position or at benzo ring of the 3-benzylideneindolin-2-one.
To determine if these compounds were E or Z isomers, the chemical shifts of the aromatic protons
on the benzylidene ring (H-2′ and H-6′) were analyzed. For the Z but not E isomer, the H-2′ or H-6′
protons would be shifted downfield due to deshielding by the carbonyl group. In the literature,
chemical shifts of 7.45-7.84 ppm to the E isomer, and 7.85-8.53 ppm were assigned to the Z isomer
[45, 48, 49]. For compounds already reported to be E isomers and whose chemical shifts were
available from the literature, comparisons of our experimental values with reported values provided
the stereochemical conformation. For compounds whose stereochemistries have not been previously
assigned, the chemical shifts of the H-2′ and H-6′ protons in the molecules were used to conclude
the configuration. The 2D NOE analysis was performed to determine the ratio of Z and E isomers.
The Z-configured compounds showed a NOE effect between the protons at the C-4 position and
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vinyl proton of the 3-benzylideneindolin-2-ones [45, 48, 49]. The ratio of Z/E was determined by
the integrals between the identified vinyl protons of Z and E isomers. The configuration and ratio of
Z/E of these compounds were summarized in Table 4.
Furthermore, to check the involvement of the substituents on the lactam nitrogen atom in the
potency and selectivity, we synthesized a group of N-substituted α-methylene-γ-lactams. The choice
of substituents was guided by the Craig plot to ensure that groups of different lipohilicities and
electronic effects were adequately represented. Starting from the optimized compound Py25,
alkylation by halide using sodium hydride as a base in DMF solution afforded smoothly the
corresponding alkylated products in quantitative yield [50] (Scheme 5). Ultimately, we synthesized
N-substituted analogues La1-3 with mere exocyclic methylene without substitution in an attempt to
assess the significance of the benzylidene substituents. These N-substituted analogues, however,
were not readily obtained by direct alkylation of α-methylene-γ-lactam due to the observable
absence of stability in the strong base solution. Alternatively, we first prepared the Horner–
Wadsworth–Emmons intermediates 12 by alkylation of 2-pyrrolidone followed by treatment of 11
with lithium diisopropylamide (LDA) and subsequent addition of diethyl chlorophosphate [51].
Then olefination of the formaldehyde using the lactams 12 as Horner–Wadsworth–Emmons
reagents in the presence of sodium hydride furnished the targeted N-substituted
α-methylene-γ-lactams La1-3 in desirable yield [52] (Scheme 6). For the 3-methyleneindolin-2-one,
the synthesis route utilized the versatile Peterson olefination of ketones via a β-silyl alcohol
intermediate which could be obtained from the reaction of indolin-2-one with
trimethylsilylmethylmagnesium chloride (Me SiCH MgCl) [53] (Scheme 4).
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Altogether, 121 final products were synthesized, analyzed and tested, of which 62 are new
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compounds not previously described in the literature. Pure compounds (≥ 95% purity) were mostly
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obtained. The structure of the synthesized compounds were confirmed by H and C NMR
spectroscopy, in addition to HPLC analysis coupled to electrospray ionization mass spectrometry
(LC/ESI-MS) which was also used to determine the purity.
2.2 Antifungal activity and structure-activity relationship study.
The α-methylene-γ-lactam (1) and (E)-3-benzylidenepyrrolidin-2-one (Py1) were chosen in the
preliminary screening to identify the efficacy of the bioisosteric replacement and chain-ring
transformation. A selection of economically important plant pathogenic fungi Fusarium
graminearum Schw., C. orbiculare, Botrytis cinerea, Fusarium oxysporum, and C. gloeosporioides
Penz. were screened. The results were summarized in Table 1. As shown, tulipalin A and compound
-1
1 were active against nearly five fungi to various extents at 50 µg mL . For the C. orbiculare, the
lactam was slightly more potent than tulipalin A with inhibition ratios of 45.7% and 34.3%,
respectively. Particularly, a similar trend in inhibitory activity was observed for these two analogues,
which could be attributed to the bioisosteric effect that the bioisosteres may elicit a similar
biological effect. In contrast, the transformational compound Py1 displayed a superior activity to its
acyclic analogue N-propylcinnamamide, which could be plausibly ascribed to the intrinsic
biological feature of α-methylene-γ-lactams and deleterious flexibility of chain conformation of the
cinnamamide derivatives. Therefore, these results validated our initial design strategy. Considering
the high efficiency in activities of these two lactam prototype compounds 1 and Py1 on C.
orbiculare among the five tested fungi, the following screening was mainly focused on the C.
orbiculare.
With the identification of efficacy of the bioisosteric replacement and chain-ring
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transformation, we continuously explored the optimal phenyl ring substitution of Py1 for the
antifungal activity against C. orbiculare. The antifungal activities were expressed as IC (50%
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inhibition concentration) and the results were shown in Table 2. Initial attempts to introduce
electron-donating CH at the para-, meta-, and ortho-position of the phenyl group of Py1, led to the
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conclusion that the para-CH substitution is better, as it can be seen that compound Py2 showed an
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improved IC of 44.7 µM. Meanwhile, the bulky group with branching chains at para-position
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conferred a dramatic potency decrease, while the CH CH group restored modest antifungal activity
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compared with unsubstituted Py1. These results showed it essential for potency to possess a smaller
alkyl group other than a branched group with larger volume and shape, which may interfere with
receptor binding. The following introduction of electron-donation CH O group on the para-position
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led to a ~2-fold increase in activity for compound Py8. When the CH O was shifted from the
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para-position to the ortho- or meta-position, it triggered the identification of a potent ortho-OCH3
compound Py10 with IC of 77.5 µM but an impotent compound Py9. With the identification of
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analogues Py8 and Py10, further SAR exploration was extensively carried out by replacing the
CH O group with more bulky CH CH O, CH (CH ) O, (CH ) CH(CH ) O, CH (CH ) O,
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C H CH O groups. Up to a four-carbon O-alkyl chain the activity level was retained. Particularly,
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the benzyl analogue Py20 manifested a ~2-fold improved activity over Py10. Further increasing the
chain length to five and twelve resulted in drastic loss of activity. Due to the ortho-alkoxyl
substitution compounds exhibited higher potency than their para-analogues, the ortho-hydroxyl
analogue Py22 was prepared and its antifungal activity was evaluated. Somewhat unexpectedly, a
considerable drop in potency was observed, despite the small size of the hydroxyl group and its
ability to form hydrogen bonding. This is probably due to the decrease in cell penetration. Further
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SAR studies of electron-withdrawing groups on the phenyl ring were performed. Substitution at the
ortho-position was strongly favored over the corresponding meta- and/or para-position as was
confirmed in the high potent compounds Py25 and Py30. While substitution of halogen atoms F, Cl
and Br at other positions retained modest activity, lipophilic group -CF was much less active. Of
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particular interest is the result obtained with Py29, which showed ~5-fold improved activity
compared to the Py1. Additionally, we explored whether incorporating simultaneously two
favorable substituents would provide synergistic effect. We observed that the disubstituted
compounds Py34 and Py35 were less potent than the related monosubstituted analogue Py30.
Finally, two bulky aryl groups, i.e., 9'-anthryl (Py36) and 2'-naphthyl (Py37), were introduced into
the exo-methylene of lactam ring in replacement of the phenyl group. The obtained compound Py36
and Py37 possessed a loss in activity (IC > 250 µM). Overall, these results not only demonstrated
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that the ortho-position of the phenyl ring of Py1 could tolerate both electron-donating and
withdrawing groups much better than the para- or meta-position, but also confirmed the importance
of substituents on the ortho-position with proper size or the electron-withdrawing characteristic.
Progressively, SAR studies to probe the core size of lactam ring were pursued by preparing a
series of α-alkenyl-δ-lactams. As can be seen from Table 3, most of the analogues showed a
significant loss of potency (IC > 250 µM). Although possessing the highest antifungal activity in
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this series of compounds, Pi17 exhibited a remarkable drop in potency in contrast to its γ-lactam
analogue Py20. Taken together, it could be supposed that the γ-lactam ring allows molecules to
adopt conformations that more closely complement the topography of receptor binding sites, while
the δ-lactam ring leads to a disruption of the bioactive conformation.
Our attention then turned to investigate the influence of the γ-lactam ring conformation on the
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potency. Taking advantage of the natural oxindole scaffold occurring a fully planar orientation of
γ-lactam ring, a collection of 3-benzylideneindolin-2-ones with different substituents on the
benzylidene ring were synthesized. Among these compounds, many of them were generally more
potent than their corresponding γ-lactam analogues. It is tempting to attribute this profile to the
increase in size, lipophilicity and rigidity of these compounds compared with the γ-lactam
analogues due to the introduction of the benzo group. Nevertheless, these compounds with different
substituents on the benzylidene ring still beared marked differences in antifungal activity where a
number of interesting SAR trends could be obtained. A moderate deterioration of activity was
observed for the electron-donating substituents CH , CH O, and CH CH O when they were
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installed into the ortho-, meta-, or para-position of the phenyl ring. Among these positions, activity
varied to a small extent, suggesting that the three groups are not indispensable for the activity.
Moreover, the bulky substituents isopropyl and tert-butyl were unfavorable to the activity. In the
case of Ox25-28, the hydroxyl substitution resulted in a significant loss of inhibitory activity,
suggesting that the hydroxyl group is disfavored. In contrast, the electron-withdrawing groups were
instrumental in improving the potency. Particularly, Ox18 with an electron-withdrawing group Cl
proved to be the most potent compound. Accordingly, this SAR trend indicated a preference for
electron-withdrawing group on the benzylidene ring of 3-benzylideneindolin-2-ones. By contrast to
such a preference, disubstitution of the benzylidene ring with two halogens did not improve activity,
as seen from a comparison of Ox23-24 and Ox17-19. With meta-Cl group consistent on
benzylidene ring, the indoline ring was decorated with different substituents including F (Ox29), Cl
(Ox30), Br (Ox31), CH (Ox32) to assess the impact of different substitutions on efficacy of the
3
efficiency 3-benzylideneindolin-2-one scaffold. No obvious increase in activity was observed for
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compounds Ox29 and Ox32 but a clear decrease for Ox30 and Ox31. A plausible explanation on
such decreases is that the Z isomers existed and accounted for a considerable proportion in the Z/E
mixtures (Z%, 65 in Ox30, 73 in Ox31). Ultimately, 3-methyleneindolin-2-one showed an improved
activity than α-methylene-γ-lactam (1) and 3-benzylideneindolin-2-one Ox1.
Before the next exploration, we performed the cytotoxicity experiments (vide infra) for the
potent compounds in different classes with the hope of pursuing a compound with desirable
selectivity in the following optimization. Compound Py25 was preferable since its high activity and
low cytotoxicity against HepG2 cells and human hepatic L02 cells. The nitrogen atom of Py25 was
modified by alkyl groups with different chain length ranging from 4 to 16. Among these analogues,
Py38 presented a ~1.5-fold decrease in activity while others suffered a significant loss. In addition,
the benzyl substitution led to a more potent compound Py43, with IC value of 38.2 µM. Then, we
5
0
varied the substitution around the benzene ring using different commercially available benzyl
bromides. Consequently, 2, 6-dichlorobenzylbromide provided the most potent analogue Py51 (IC50
= 10.4 µM) in our hand. Further comparisons between the N-substituted α-methylene-γ-lactams
(La1-3) and their corresponding N-substituted Py25 analogues were performed. There was no
apparent increase in activity for N-substituted α-methylene-γ-lactams, shedding light on the role of
the phenyl groups on the exo-methylene of the γ-lactam scaffold.
2.3 Cytotoxicity.
In the process of scaffold optimization, the cytotoxicity and selectivity were comprehensively
considered. The cytotoxicity of the highly potent compounds in each class was assessed evaluated
in HepG2 cells and human hepatic L02 cells. Either the N-unsubstituted or the N-substituted
α-arylidene-γ-lactams were generally less toxic to HepG2 cells and human hepatic L02 cells,
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emphasizing a good selectivity. This finding was in agreement with the previous report for
α-arylidene-γ-lactones where induction of the appropriate substituents on the α-arylidene group
could reduce their toxicity [34-36]. For the α-arylidene-δ-lactams, their cytotoxicity is as
comparative
as
their antifungal
activity while being less
efficient.
Albeit
3-benzylideneindolin-2-ones were equipped with high antifungal activity, their nonnegligible
cytotoxicity led to a poor selectivity. Beside the HepG2 cells and human hepatic L02 cells,
3-benzylideneindolin-2-ones had reported cytotoxicity against human cancer cell lines MCF-7 and
HCT116 [48]. Summarizing, the N-benzyl substituted analogues possessed good selectivity together
with excellent antifungal activity, suggesting a preferable natural-based scaffold for further lead
optimization.
2.4 The ultrastructural change studies.
In the primary infection of diseases cycle, the germinated spores produced the germ tube
which might improve the adherence to host cells and penetration into the host. Therefore, blocking
the spore germination and germ tube growth has a pivotal role in the control of plant diseases. Apart
from inhibition of spore germination, compound Py51 was observed to inhibit the germ tube growth.
TEM was used to determine ultrastructural changes of germ tubes induced by Py51. The results
may lay a foundation for the further antifungal mechanism studies on this natural-based scaffold.
Control cells were featured with the well-defined cytoplasmic integrity intact (Fig. 4 A, B).
The visible cytoplasmic membrane showed good adhesion to cell wall that appeared as a bright,
fibrous layer. Various identifiable organelles distinct in their constituent elements, and normal size
vacuoles were observed in cells. The cell nuclei surrounded by the nuclear membranes, and
electron-dense nucleoli were visible in many cases. The numerous mitochondria with membranous
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cristae were visibly presented. Many of them were located in the apical region of growing germ
tubes to supply energy for elongation, indicating that the cells were growing rapidly.
Compared with the control, the treated cells revealed an abnormal subcellular morphology (Fig.
4 C-F). After 12h of treatment, the fungal cells have already showed some alternations of the inner
membranes that became irregular in thickness and density. Although internal organelles like
mitochondria were well preserved, they witnessed an aberrant morphology characterized by an
abnormality in the size of individual mitochondria and a concomitant increase in the number.
Vacuoles swelled in irregular shape. After 24h of treatment, many cells were damaged, showing
thickening cell wall, cytoplasm membrane detachment integratedly from cell wall, invaginations of
cytoplasm membrane, occurrence of numerous vacuoles, and mitochondrial membrane
discontinuity or disappearance. When treatment was prolonged up to 48h, cytoplasm was clearly
degenerated due to the high vacuolization and organelles were not recognized any more. At 72h, a
large part of the fungal structures was disrupted, and many cells looked empty.
Taken together, a remarkable ultrastructural change was observed for mitochondria
encompassing the abnormality in the size and number of mitochondria in 12h and mitochondrial
membrane discontinuity or disappearance in 24h. As is known, mitochondria perform pivotal
functions essential for ATP production, homeostasis, and metabolism. The alternations in the size
and number of mitochondria might be subjected to the disruption of mitochondrial functions during
the early stage. After a long period of treatment, the mitochondria suffered a seriously disruptive
damage, leading to the membrane discontinuity or disappearance. These results revealed that the
mitochondria appeared one of the primary targets.
3. Conclusion
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In this study, 62 new and 59 known natural α-methylenelactam analogues including
α-methylene-γ-lactams, α-arylidene-γ and δ-lactams, and 3-arylideneindolin-2-ones were
synthesized and investigated for their antifungal activity. Bioassay results indicated that most of the
α-arylidene-γ-lactams possessed superior activity over the α-arylidene-γ-lactams and better
selectivity than 3-arylideneindolin-2-ones. Compound Py51 possessed the highest antifungal
activity against C. orbiculare with IC = 10.4 µM but poor cytotoxic activity with IC = 141.2 µM
5
0
50
(against HepG2 cell line) and 161.2 µM (against human hepatic L02 cell line). Ultrastructural
changes studies showed that Py51 could cause important cell morphological changes in C.
orbiculare, such as plasma membrane detached from cell wall, cell wall thickening, mitochondria
disruption, a dramatic increase in vacuolation, and eventually a complete loss in the integrity of
organelles. Significantly, mitochondria appeared one of the primary targets, as confirmed by their
remarkably aberrant morphological changes. Further structural optimization is still well ongoing
with the aim of discovering and developing improved natural-based crop-protection agents.
4. Experimental section
4.1. General.
All the starting materials, reagents, and solvents were obtained from commercial sources and
used without further purification unless otherwise stated. The reaction solvents were distilled prior
to use. Tetrahydrofuran (THF), diethyl ether (Et O) and toluene were distilled from sodium and
2
benzophenone under an Ar atmosphere. Dimethylformamide (DMF), triethylamine(Et N),
3
diisopropylamine and dichloromethane (DCM) were distilled from calcium hydride and stored in
4Å molecular sieves. Generally, all reactions were carried out using standard air-free and
moisture-free techniques under an inert nitrogen atmosphere with dry solvents unless otherwise
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stated. Reaction progress was monitored by thin layer chromatography (TLC) using GF254 silica
gel with fluorescent indicator on glass plates. Visualization was achieved with UV light and
Dragendorff's reagent unless otherwise stated. Chromatography was performed using Qingdao
1
13
Haiyang silica gel (200–300 mesh). H and C NMR spectra were recorded at ambient temperature
1
13
1
on a Bruker AMX500 ( H at 500 MHz, C at 125 MHz) magnetic resonance spectrometer. H
chemical shifts were reported in ppm using residual CHCl (δ 7.26) and DMSO-d (δ 2.50) as
3
6
internal standards. Spin multiplicities are described as br (broad singlet), s (singlet), d (doublet), t
(triplet), q (quartet), and m (multiplet). Coupling constants (J) were reported in Hertz (Hz). Proton
1
3
decoupled C NMR spectra were reported in ppm (δ) relative to residual CHCl (δ 77.0) and
3
DMSO (39.5).
4.2. Synthesis.
4.2.1. General procedure for synthesis of N-propylcinnamamide. [54]
Propan-1-amine (0.9 eq) and 4-dimethylaminopyridine (DMAP) (1.0 eq) were added to a
stirred solution of cinnamic acid (1.0 eq) in anhydrous DCM. After the reaction mixture was cooled
in an ice bath, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCóHCl) (1.0 eq)
was added. After stirring for 24 h, the reaction mixture was washed with a saturated brine solution
and water. The residual organic layer was dried by Na SO and then purified by silica gel
2
4
chromatography and eluted with petroleum ether/EtOAc (10/3) to yield pure N-propylcinnamamide
o
1
as a white solid. Yield: 85%; m.p.: 82-83 C; H NMR (500 MHz, CDCl ) δ (ppm): 7.61 (d, J =
3
H
15.6 Hz, 1H), 7.44-7.46 (m, 2H), 7.29-7.30 (m, 3H), 6.48 (d, J = 15.7 Hz, 1H), 6.32 (br, 1H),
1
3
3.32-3.36 (m, 2H), 1.55-1.62 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H); C NMR (125 MHz, CDCl ) δ
3
C
(ppm): 166.2, 140.6, 135.0, 129.5, 128.8, 127.7, 121.2, 41.5, 22.9, 11.5; ESI-MS: m/z 212.1
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([M+Na] ).
4.2.2. General procedure for synthesis of α-methylene-γ-lactam (1). [39, 40]
N-boc-2-pyrrolidone. To a solution of 2-pyrrolidinone (8.51 g, 100 mmol, 1.0 eq) in DCM
(200 mL) were added DMAP (12.2 g, 100 mmol, 1.0 eq), Et N (13.8 mL, 100 mmol, 1.0 eq),
3
di-tert-butyl dicarbonate (Boc O) (43.65 g, 200 mmol, 2.0 eq). CO was released from the stirring
2
2
solution. The reaction mixture was stirred at room temperature for 12 h. The solvent was then
evaporated under reduced pressure and the residual product was purified by silica gel
chromatography (petroleum ether/EtOAc = 4/1) to afford N-boc-2-pyrrolidone as a white solid.
1
Yield: 86%; H NMR (500 MHz, CDCl ) δ (ppm): 3.72 (t, J = 7.0 Hz, 2H), 2.48 (t, J = 8.2 Hz, 2H),
3
H
13
1.94-2.01 (m, 2H), 1.50 (s, 9H); C NMR (125 MHz, CDCl ) δ (ppm): 174.3, 150.2, 82.7, 46.5,
3
C
+
32.9, 28.0, 17.4; ESI-MS: m/z 208.1 ([M+Na] ).
α-Methylene-γ-lactam. Diethyl oxalate (7.3 g, 50 mmol, 2.0 eq) and sodium hydride (1.2 g, 30
mmol, 1.2 eq) were added to diethyl ether (20 mL) and stirred at 40 °C. A solution of
N-boc-2-pyrrolidone (4.6 g, 25 mmol, 1.0 eq) in 20 mL diethyl ether was added dropwise over a
period of 1h, and the mixture was stirred at 40 °C for 2 days. After that, the sodium salt was filtered
from the resulting yellow solution, washed with diethyl ether and redissolved in DMF.
Paraformaldehyde (1.5 g, 50 mmol, 2.0 eq) was added and the reaction mixture was heated to
100 °C for 1.5 h. After cooling, the reaction mixture was filtered, dried and concentrated under
reduced pressure to give a yellow oil residue. The above oil product (2.0 g, 10 mmol) was added to
a stirred DCM solution (20 mL) at room temperature. Trifluoroacetic acid (2 mL) was added to the
solution and the stirring was continued for 0.5 h. Finally, the reaction mixture was concentrated,
which was followed by addition of DCM (20 mL) and concentration (× 4). Purification was
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subsequently performed by column chromatography on silica gel, yielding α-methylene-γ-lactam.
1
Yield: 16%; H NMR (500 MHz, CDCl ) δ (ppm): 6.03 (t, J = 2.8 Hz, 1H), 5.45 (t, J = 2.5 Hz, 1H),
3
H
1
3
3.44-3.50 (m, 2H), 2.77-2.84 (m, 2H); C NMR (125 MHz, CDCl ) δ (ppm): 167.9, 139.6, 115.7,
3
C
+
43.5, 23.9. ESI-MS: m/z 98.1 ([M+H] ).
4.2.3 General procedure for synthesis of α-alkenyl-γ-lactams (Py1-40) and α-alkenyl-δ-lactams
(Pi1-32). [43]
2-Pyrrolidinone or 2-piperidinone (11 mmol, 1.1 eq) was dissolved in dry toluene (5 ml). To
this solution, trifluoroacetic anhydride (TFAA) (2.5g, 12 mmol, 1.2eq) was charged slowly by a
syringe at 5 °C. Upon complete addition, the solution was stirred for 1h at room temperature.
Toluene was removed under vacuum using a rotary evaporator. After evaporation, another portion
of toluene (10 ml) was added to the concentrate and again removed under vacuum, which was
repeated twice. The resulting pale yellow liquid was then mixed with corresponding substituted
benzaldehyde (10 mmol, 1.0 eq) and the homogeneous solution in a syringe was slowly injected to a
1M solution of potassium t-butoxide (KO Bu) in THF (13 ml, 13 mmol, 1.3 eq) at 0 °C. Once
t
complete addition, the mixture was heated to 55 °C and stirred therein for 1 h. Then the reaction
mixture was concentrated to a thick slurry. To the concentrate with vigorous stirring, water (100 ml)
was added slowly to precipitate the crude product. The water solution with precipitate was cooled to
5 °C and held therein for 1 h. The solution was filtered, and the solid was washed with water, and
dried in vacuum oven overnight. The dried crude product was purified by recrystallization from
petroleum ether/EtOAc.
(E)-3-benzylidenepyrrolidin-2-one (Py1) [55]: Light yellow solid; yield: 45%; mp:
1
102-103 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.14 (br, 1H), 7.32-7.54 (m, 5H), 7.09 (t, J
6
H
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0

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= 2.5 Hz, 1H), 3.36 (t, J = 6.5 Hz, 2H), 3.06 (td, J = 6.3, 2.4 Hz, 2H); C NMR (125 MHz,
13
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
DMSO-d ) δ (ppm): 170.5, 135.6, 132.6, 129.2, 128.7, 128.2, 127.7, 38.7, 25.8; HR-MS (ESI): m/z
6
C
+
calcd for C H NONa ([M+Na] ) 196.0733, found 196.0735.
11
11
(E)-3-(4-methylbenzylidene)pyrrolidin-2-one (Py2) [56]: White solid; yield: 47%; mp:
1
104-105 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.08 (br, 1H), 7.23-7.42 (m, 4H), 7.05 (t, J
6
H
1
3
= 2.5 Hz, 1H), 3.36 (t, J = 6.5 Hz, 2H), 3.03 (td, J = 6.3, 2.4 Hz, 2H), 2.32 (s, 3H); C NMR (125
MHz, DMSO-d ) δ (ppm): 170.6, 137.8, 132.8, 131.4, 129.3, 129.2, 127.6, 38.7, 25.8, 20.8;
6
C
+
HR-MS (ESI): m/z calcd for C H NONa ([M+Na] ) 210.0889, found 210.0893.
1
2
13
(E)-3-(3-methylbenzylidene)pyrrolidin-2-one (Py3): Light yellow solid; yield: 37%; mp:
1
101-102 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.10 (br, 1H), 7.15-7.33 (m, 4H), 7.04 (t, J
6
H
1
3
= 2.8 Hz, 1H), 3.36 (t, J = 6.2 Hz, 2H), 3.03 (td, J = 6.5, 2.7 Hz, 2H), 2.33 (s, 3H); C NMR (125
MHz, DMSO-d ) δ (ppm): 170.5, 137.8, 135.5, 132.3, 129.8, 128.9, 128.5, 127.7, 126.3, 38.6, 25.8,
6
C
+
20.9; HR-MS (ESI): m/z calcd for C H NONa ([M+Na] ) 210.0889, found 210.0893.
1
2
13
(E)-3-(2-methylbenzylidene)pyrrolidin-2-one (Py4): Light yellow solid; yield: 35%; mp:
1
104-105 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.13 (br, 1H), 7.23-7.47 (m, 5H), 3.33 (t, J
6
H
1
3
= 6.5 Hz, 2H), 2.97 (td, J = 6.8, 2.8 Hz, 2H), 2.32 (s, 3H); C NMR (125 MHz, DMSO-d ) δ
6
C
(ppm): 170.4, 136.9, 134.2, 133.1, 130.2, 128.0, 127.6, 125.9, 125.1, 38.7, 25.5, 19.4; HR-MS (ESI):
+
m/z calcd for C H NONa ([M+Na] ) 210.0889, found 210.0893.
1
2
13
(E)-3-(4-ethylbenzylidene)pyrrolidin-2-one (Py5): White solid; yield: 49%; mp: 109-111 °C;
H NMR (500 MHz, DMSO-d ) δ (ppm): 8.09 (br, 1H), 7.26-7.45 (m, 4H), 7.05 (t, J = 2.5 Hz, 1H),
1
6
H
3.36 (t, J = 6.5 Hz, 2H), 3.03 (td, J = 6.4, 2.4 Hz, 2H), 2.62 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz,
1
3
3H); C NMR (125 MHz, DMSO-d ) δ (ppm): 170.6, 144.1, 133.2, 131.5, 129.3, 128.1, 127.6,
6
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38.7, 27.9, 25.7, 15.4; HR-MS (ESI): m/z calcd for C H NONa ([M+Na] ) 224.1046, found
13 15
+
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
224.1049.
(E)-3-(4-isopropylbenzylidene)pyrrolidin-2-one (Py6): White solid; yield: 43%; mp:
1
113-115 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.08 (br, 1H), 7.29-7.46 (m, 4H), 7.05 (t, J =
6
H
2.6 Hz, 1H), 3.36 (t, J = 6.4 Hz, 2H), 3.03 (td, J = 6.6, 2.4 Hz, 2H), 2.86-2.94 (m, 1H), 1.20 (d, J =
1
3
6.9 Hz, 6H); C NMR (125 MHz, DMSO-d ) δ (ppm): 170.6, 148.6, 133.2, 131.5, 129.3, 127.6,
6
C
+
126.6, 38.6, 33.2, 25.7, 23.6; HR-MS (ESI): m/z calcd for C H NONa ([M+Na] ) 238.1202, found
1
4
17
238.1205.
(E)-3-(4-(tert-butyl)benzylidene)pyrrolidin-2-one (Py7): White solid; yield: 47%; mp:
1
112-113 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.10 (br, 1H), 7.43-7.47 (m, 4H), 7.06 (t, J =
6
H
13
2.9 Hz, 1H), 3.36 (t, J = 6.4 Hz, 2H), 3.03 (td, J = 6.5, 2.8 Hz, 2H), 1.28 (s, 9H); C NMR (125
MHz, DMSO-d ) δ (ppm): 171.1, 151.3, 133.3, 132.1, 129.6, 128.0, 126.0, 39.2, 34.9, 31.4, 26.2;
6
C
+
HR-MS (ESI): m/z calcd for C H NONa ([M+Na] ) 252.1359, found 252.1361.
1
5
19
(E)-3-(4-methoxybenzylidene)pyrrolidin-2-one (Py8) [56]: Light yellow solid; yield: 51%;
1
mp: 121-122 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.02 (br, 1H), 7.48 (d, J = 8.8 Hz, 2H),
6
H
7.03 (t, J = 2.7 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 3.78 (s, 3H), 3.36 (t, J = 6.4 Hz, 2H), 3.02 (td, J =
1
3
6.5, 2.4 Hz, 2H); C NMR (125 MHz, DMSO-d ) δ (ppm): 170.7, 159.2, 130.8, 129.7, 128.2,
6
C
+
127.4, 114.2, 55.1, 38.6, 25.7; HR-MS (ESI): m/z calcd for C H NO Na ([M+Na] ) 226.0838,
1
2
13
2
found 226.0839.
(E)-3-(3-methoxybenzylidene)pyrrolidin-2-one (Py9): Light yellow solid; yield: 31%; mp:
1
119-121 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.12 (br, 1H), 7.34 (m, 1H), 6.91-7.11 (m,
6
H
1
3
4H), 3.78 (s, 3H), 3.36 (t, J = 6.2 Hz, 2H), 3.06 (td, J = 6.5, 2.7 Hz, 2H); C NMR (125 MHz,
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506
DMSO-d ) δ (ppm): 170.9, 159.8, 137.5, 133.4, 130.2, 128.2, 122.1, 115.0, 114.5, 55.5, 39.2, 26.2;
6
C
+
HR-MS (ESI): m/z calcd for C H NO Na ([M+Na] ) 226.0838, found 226.0839.
1
2
13
2
(E)-3-(2-methoxybenzylidene)pyrrolidin-2-one (Py10): Light yellow solid; yield: 34%; mp:
1
117-118 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.07 (br, 1H), 7.31-7.48(m, 3H), 7.05 (d, J =
6
H
8.3 Hz, 1H), 7.00 (t, J = 7.5 Hz, 1H), 3.83 (s, 3H), 3.33 (t, J = 6.3 Hz, 2H), 2.99 (td, J = 6.5, 2.7 Hz,
1
3
2H); C NMR (125 MHz, DMSO-d ) δ (ppm): 170.9, 159.8, 137.5, 133.4, 130.2, 128.2, 122.1,
6
C
+
115.0, 114.5, 55.5, 39.2, 26.2; HR-MS (ESI): m/z calcd for C H NO Na ([M+Na] ) 226.0838,
1
2
13
2
found 226.0839.
(E)-3-(4-ethoxybenzylidene)pyrrolidin-2-one (Py11) [57]: White solid; yield: 48%; mp:
1
141-144 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.03 (br, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.03
6
H
(t, J = 2.7 Hz, 1H), 6.97 (d, J = 8.6 Hz, 2H), 4.04 (q, J = 7.0 Hz, 2H), 3.35 (t, J = 6.2 Hz, 2H), 3.00
13
(td, J = 7.0, 2.5 Hz, 2H), 1.32 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, DMSO-d ) δ (ppm): 171.3,
6
C
159.0, 131.3, 130.1, 128.6, 128.0, 115.1, 36.6, 39.1, 26.1, 15.0; HR-MS (ESI): m/z calcd for
+
C H NO Na ([M+Na] ) 240.0995, found 240.0995.
13
15
2
(E)-3-(2-ethoxybenzylidene)pyrrolidin-2-one (Py12): Light yellow solid; yield: 38%; mp:
1
134-136 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.09 (br, 1H), 7.28-7.48(m, 3H), 7.03 (d, J =
6
H
8.5 Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 4.07 (q, J = 6.7 Hz, 2H), 3.33 (t, J = 6.4 Hz, 2H), 2.99 (td, J =
1
3
6.3, 2.7 Hz, 2H), 1.32 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, DMSO-d ) δ (ppm): 171.2, 157.1,
6
C
132.5, 130.2, 129.0, 124.7, 122.6, 120.7, 112.5, 64.0, 39.2, 26.3, 15.1; HR-MS (ESI): m/z calcd for
+
C H NO Na ([M+Na] ) 240.0995, found 240.0995.
13
15
2
4-Butoxybenzaldehyde (4a) [58]: In a 100 mL round bottomed flask equipped with a reflux
condenser and a magnetic stirring bar were dissolved 4-hydroxybenzaldehyde (35 g, 287 mmol) and
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1-bromobutane (30.92 ml, 287 mmol) in DMF (750 mL). The mixture then was stirred under
nitrogen for 20 minutes. Anhydrous potassium carbonate (118.83 g, 860 mmol) was then added and
the mixture was stirred and heated at 70 °C under nitrogen. After 20 h, the reaction was allowed to
cool to room temperature, treated with excess water, and extracted with ethyl acetate. The combined
organic extracts were then washed with water several times and dried over anhydrous sodium
sulfate, filtered. The crude product was purified by chromatography (PE/EA, 20/1) yielding a brown
1
oil. Yield: 95%; H NMR (500 MHz, CDCl ) δ (ppm): 9.89 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.00
3
H
(d, J = 8.6 Hz, 2H), 4.06 (t, J = 6.3 Hz, 2H), 1.79-1.85 (m, 2H), 1.49-1.57 (m, 2H), 1.01 (t, J = 7.3
1
3
Hz, 3H); C NMR (125 MHz, CDCl ) δ (ppm): 190.7, 164.2, 131.9, 129.8, 114.7, 68.1, 31.0, 19.1,
3
C
+
13.7; ESI-MS: m/z 179.2 ([M+H ]); The following alkoxy benzaldehyde derivatives 4b-h were
synthesized in a similar method.
(E)-3-(4-butoxybenzylidene)pyrrolidin-2-one (Py13): White solid; yield: 41%; mp:
1
151-153 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.00 (br, 1H), 7.45 (d, J = 7.9 Hz, 2H),
6
H
6.97-7.03 (m. 3H), 3.99 (t, J = 6.3 Hz, 2H), 3.35 (t, J = 6.2 Hz, 2H), 3.01 (td, J = 7.0, 2.5 Hz, 2H),
1
3
1.67-1.70 (m, 2H), 1.41-1.44 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H); C NMR (125 MHz, DMSO-d ) δ
6
C
(ppm): 171.3, 159.2, 131.3, 130.1, 128.6, 128.0, 115.1, 67.7, 39.1, 31.1, 26.2, 19.2, 14.1; HR-MS
+
(ESI): m/z calcd for C H NO Na ([M+Na] ) 268.1308, found 268.1310.
1
5
19
2
1
2-Butoxybenzaldehyde (4b) [59]: Brown oil; yield: 91%; H NMR (500 MHz, CDCl ) δ
3
H
(ppm): 10.49 (s, 1H), 7.78-7.90 (m, 1H), 7.47-7.49 (m, 1H), 6.95-6.96 (m, 2H), 4.04 (t, J = 6.3 Hz,
1
3
2H), 1.79-1.80 (m, 2H), 1.49-1.52 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H); C NMR (125 MHz, CDCl )
3
δC (ppm): 189.7, 161.5, 135.8, 128.0, 124.8, 120.3, 112.6, 68.1, 31.0, 19.2, 13.7; ESI-MS: m/z 179.2
+
([M+H ]).
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(E)-3-(2-butoxybenzylidene)pyrrolidin-2-one (Py14): White solid; yield: 29%; mp:
1
147-148 °C; H NMR (500 MHz, CDCl ) δ (ppm): 7.78 (t, J = 2.7 Hz, 1H), 7.45-7.46 (m, 1H),
3
H
7.30-7.33 (m, 1H), 7.17 (br, 1H), 6.94-7.01 (m, 2H), 4.05 (t, J = 6.6 Hz, 2H), 3.55 (t, J = 6.4 Hz,
2H), 3.12 (td, J = 6.5, 2.6 Hz, 2H), 1.83-1.89 (m, 2H), 1.51-1.58 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H);
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C NMR (125 MHz, CDCl ) δ (ppm): 157.6, 130.2, 129.8, 128.9, 125.2, 125.0, 120.0, 111.9, 68.2,
3
C
+
39.7, 31.2, 26.6, 19.3, 13.8; HR-MS (ESI): m/z calcd for C H NO Na ([M+Na] ) 268.1308, found
1
5
19
2
268.1310.
1
4-(Isopentyloxy)benzaldehyde (4c) [60]: Brown oil; yield: 92%; H NMR (500 MHz, CDCl )
3
δH (ppm): 9.89 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 4.09 (t, J = 6.4 Hz, 2H),
1
3
1.82-1.90 (m, 1H), 1.70-1.75 (m, 2H), 1.00 (d, J = 6.7 Hz, 6H); C NMR (125 MHz, CDCl ) δ
3
C
+
(ppm): 190.7, 164.2, 131.9, 129.8, 114.7, 66.8, 37.7, 25.0, 22.5; ESI-MS: m/z 193.3 ([M+H ]).
(E)-3-(4-(isopentyloxy)benzylidene)pyrrolidin-2-one (Py15): White solid; yield: 54%; mp:
1
141-142 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.00 (br, 1H), 7.45 (d, J = 7.9 Hz, 2H),
6
H
6.98-7.03 (m. 3H), 4.02 (t, J = 6.3 Hz, 2H), 3.35 (t, J = 6.2 Hz, 2H), 3.01 (td, J = 7.0, 2.5 Hz, 2H),
1
3
1.73-1.80 (m, 1H), 1.59-1.63 (m, 2H), 0.92 (d, J = 6.6 Hz, 6H); C NMR (125 MHz, DMSO-d ) δ
6
C
(ppm):159.1, 131.3, 130.1, 128.6, 128.0, 115.2, 66.4, 39.1, 37.8, 26.1, 25.0, 22.9; HR-MS (ESI):
+
m/z calcd for C H NO Na ([M+Na] ) 282.1465, found 282.1466.
1
6
21
2
1
2-(Isopentyloxy)benzaldehyde (4d) [59]: Brown oil; yield: 98%; H NMR (500 MHz, CDCl )
3
δH (ppm): 10.54 (s, 1H), 7.85-7.86 (m, 1H), 7.54-7.57 (m, 1H), 7.01-7.04 (m, 2H), 4.14 (t, J = 6.5
13
Hz, 2H), 1.85-1.94 (m, 1H), 1.76-1.80 (m, 2H), 1.00 (d, J = 6.7 Hz, 6H); C NMR (125 MHz,
CDCl ) δ (ppm): 189.8, 161.5, 135.9, 128.2, 124.9, 120.4, 112.5, 66.9, 37.7, 25.1, 22.5; ESI-MS:
3
C
+
m/z 193.3 ([M+H ]).
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(E)-3-(2-(isopentyloxy)benzylidene)pyrrolidin-2-one (Py16): White solid; yield: 39%; mp:
1
135-137 °C; H NMR (500 MHz, CDCl ) δ (ppm): 7.78 (t, J = 2.7 Hz, 1H), 7.45-7.46 (m, 1H),
3
H
7.30-7.33 (m, 1H), 7.07 (br, 1H), 6.95-7.01 (m, 2H), 4.07 (t, J = 6.6 Hz, 2H), 3.55 (t, J = 6.4 Hz,
2H), 3.12 (td, J = 6.5, 2.6 Hz, 2H), 1.85-1.93 (m, 1H), 1.75-1.80 (m, 2H), 1.00 (d, J = 6.6 Hz, 6H);
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C NMR (125 MHz, CDCl ) δ (ppm): 172.8, 157.6, 130.1, 129.8, 128.9, 125.3, 125.0, 120.0,
3
C
+
111.8, 67.0, 39.7, 37.9, 26.6, 25.1, 22.6; HR-MS (ESI): m/z calcd for C H NO Na ([M+Na] )
16
21
2
282.1465, found 282.1466.
1
4-(Dodecyloxy)benzaldehyde (4e) [61]: Brown oil; yield: 97%; H NMR (500 MHz, CDCl )
3
δH (ppm): 9.89 (s, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 4.05 (t, J = 6.6 Hz, 2H),
1
3
1.80-1.86 (m, 2H), 1.45-1.51 (m, 2H), 1.29-1.39 (m, 16H), 0.90 (t, J = 7.0 Hz, 3H); C NMR (125
MHz, CDCl ) δ (ppm): 190.6, 164.2, 131.9, 129.8, 114.7, 68.4, 31.9, 29.6, 29.6, 29.5, 29.5, 29.3,
3
C
+
29.0, 25.9, 22.6, 14.1; ESI-MS: m/z 291.4 ([M+H ]).
(E)-3-(4-(dodecyloxy)benzylidene)pyrrolidin-2-one (Py17): White solid; yield: 42%; mp:
1
165-167 °C; H NMR (500 MHz, CDCl ) δ (ppm): 7.46 (d, J = 6.7 Hz, 2H), 7.34 (t, J = 2.9 Hz,
3
H
1H), 6.96 (d, J = 8.6 Hz, 2H), 4.02 (t, J = 6.5 Hz, 2H), 3.58 (t, J = 6.6 Hz, 2H), 3.17 (td, J = 7.0, 2.5
1
3
Hz, 2H), 1.79-1.85 (m, 2H), 1.46-1.52 (m, 2H), 1.29-1.39 (m, 16H), 0.91 (t, J = 7.0 Hz, 3H); C
NMR (125 MHz, CDCl ) δ (ppm): 159.5, 131.1, 130.2, 128.2, 127.1, 114.7, 68.1, 39.5, 31.9, 29.6,
3
C
29.6, 29.6, 29.5, 29.3, 29.3, 29.2, 26.3, 26.0, 22.7; HR-MS (ESI): m/z calcd for C H NO Na
2
3
35
2
+
([M+Na] ) 380.2560, found 380.2564.
1
2-(Dodecyloxy)benzaldehyde (4f) [62]: Brown oil; yield: 93%; H NMR (500 MHz, CDCl )
3
δH (ppm): 10.5 (s, 1H), 7.83-7.85 (m, 1H), 7.52-7.55 (m, 1H), 6.98-7.02 (m, 2H), 4.09 (t, J = 6.4 Hz,
1
3
2H), 1.80-1.86 (m, 2H), 1.48-1.52 (m, 2H), 1.29-1.39 (m, 16H), 0.91 (t, J = 7.0 Hz, 3H); C NMR
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(125 MHz, CDCl ) δ (ppm): 189.7, 161.5, 135.8, 128.1, 124.9, 120.4, 112.5, 31.9, 29.6, 29.6, 29.6,
3
C
+
29.5, 29.3, 29.1, 26.0, 22.7, 14.1; ESI-MS: m/z 291.4 ([M+H ]).
(E)-3-(2-(dodecyloxy)benzylidene)pyrrolidin-2-one (Py18): White solid; yield: 36%; mp:
1
152-153 °C; H NMR (500 MHz, CDCl ) δ (ppm): 7.78 (t, J = 2.7 Hz, 1H), 7.45-7.46 (m, 1H),
3
H
7.30-7.33 (m, 1H), 6.94-7.01 (m, 2H), 4.03 (t, J = 6.6 Hz, 2H), 3.55 (t, J = 6.4 Hz, 2H), 3.12 (td, J =
6.5, 2.6 Hz, 2H), 1.84-1.89 (m, 2H), 1.46-1.52 (m, 2H), 1.30-1.38 (m, 16H), 0.92 (t, J = 7.0 Hz, 3H);
1
3
C NMR (125 MHz, CDCl ) δ (ppm): 173.0, 157.6, 130.4, 129.7, 128.9, 125.1, 125.1, 120.0,
3
C
111.9, 68.6, 39.8, 31.9, 29.6, 29.6, 29.5, 29.4, 29.3, 29.2, 26.6, 26.0, 22.7, 14.1; HR-MS (ESI): m/z
+
calcd for C H NO Na ([M+Na] ) 380.2560, found 380.2564.
2
3
35
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1
4-(Benzyloxy)benzaldehyde (4g) [63]: White solid; yield: 91%; mp: 70-71 °C; H NMR (500
MHz, CDCl ) δ (ppm): 9.93 (s, 1H), 7.87-7.89 (m, 2H), 7.38-7.49 (m, 5H), 7.11-7.13 (m, 2H), 5.19
3
H
1
3
(s, 2H); C NMR (125 MHz, CDCl ) δ (ppm): 190.7, 163.7, 136.0, 132.0, 130.2, 128.7, 128.3,
3
C
+
127.5, 115.2, 70.3; ESI-MS: m/z 213.0 ([M+H ]).
(E)-3-(4-(benzyloxy)benzylidene)pyrrolidin-2-one (Py19): White solid; yield: 49%; mp:
1
133-135 °C; H NMR (500 MHz, DMSO-d ) δ (ppm): 8.04 (br, 1H), 7.34-7.51 (m, 7H), 7.07-7.11
6
H
1
3
(m, 3H), 5.17 (s, 2H), 3.38 (t, J = 6.3 Hz, 2H), 3.04 (td, J = 6.4, 2.5 Hz, 2H); C NMR (125 MHz,
DMSO-d ) δ (ppm): 171.3, 158.8, 137.3, 131.3, 130.4, 128.9, 128.9, 128.3, 128.1, 127.9, 115.5,
6
C
+
69.7, 39.1, 26.1; HR-MS (ESI): m/z calcd for C H NO Na ([M+Na] ) 302.1151, found 302.1155.
1
8
17
2
1
2-(Benzyloxy)benzaldehyde (4h) [64]: White solid; yield: 97%; mp: 49-51 °C; H NMR (500
MHz, CDCl ) δ (ppm): 10.06 (s, 1H), 7.90-7.92 (m, 1H), 7.38-7.59 (m, 6H), 7.06-7.11 (m, 2H),
3
H
1
3
5.22 (s, 2H); C NMR (125 MHz, CDCl ) δ (ppm): 189.7, 161.1, 136.1, 135.9, 128.7, 128.4, 128.3,
3
C
+
127.3, 125.2, 121.0, 113.1, 70.5; ESI-MS: m/z 213.0 ([M+H ]).
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(E)-3-(2-(benzyloxy)benzylidene)pyrrolidin-2-one (Py20): White solid; yield: 42%; mp:
1
142-144 °C; H NMR (500 MHz, CDCl ) δ (ppm): 7.78 (t, J = 2.7 Hz, 1H), 7.27-7.49 (m, 8H),
3
H
13
6.97-7.04 (m, 2H), 5.17 (s, 2H), 3.54 (t, J = 6.3 Hz, 2H), 3.11 (td, J = 6.4, 2.5 Hz, 2H); C NMR
(125 MHz, CDCl ) δ (ppm): 172.8, 157.1, 136.9, 130.7, 129.7, 129.1, 128.6, 127.8, 127.1, 125.4,
3
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+
125.1, 120.6, 112.6, 7.03, 39.7, 26.6; HR-MS (ESI): m/z calcd for C H NO Na ([M+Na] )
18
17
2
302.1151, found 302.1155.
(E)-3-(2-((tert-butyldimethylsilyl)oxy)benzylidene)pyrrolidin-2-one
(Py21):
(2-((tert-butyldimethylsilyl)oxy)benzaldehyde was prepared according to the reported literature)
1
White solid; yield: 36%; H NMR (500 MHz, CDCl ) δ (ppm): 7.66 (s, 1H), 7.50-7.57 (m, 2H),
3
H
7.32-7.36 (m, 3H), 3.71 (t, J = 6.3 Hz, 2H), 2.93 (td, J = 6.4, 2.5 Hz, 2H), 1.02 (s, 9H), 0.24 (s, 6H);
1
3
C NMR (125 MHz, CDCl ) δ (ppm): 162.6, 153.3, 141.6, 131.6, 127.5, 125.8, 124.8, 119.0,
3
C
+
116.6, 39.3, 30.6, 25.6, 18.2, -4.3; ESI-MS: m/z 304.5 ([M+H ]).
1
(E)-3-(2-hydroxybenzylidene)pyrrolidin-2-one (Py22): White solid; yield: 42%; H NMR
(500 MHz, DMSO-d ) δ (ppm): 9.53 (br, 1H), 8.07 (br, 1H), 7.23-7.26(m, 3H), 6.95-7.02 (m, 3H),
6
H
13
6.77-6.79 (m, 1H), 3.39 (t, J = 6.4 Hz, 2H), 3.05 (td, J = 6.5, 2.6 Hz, 2H); C NMR (125 MHz,
DMSO-d ) δ (ppm): 171.0, 157.9, 137.3, 132.7, 130.1, 128.4, 120.9, 116.2, 115.9, 39.1, 26.3;
6
C
+
HR-MS (ESI): m/z calcd for C H NO Na ([M+Na] ) 212.0682, found 212.0683.
11
11
2
(E)-3-(4-(dimethylamino)benzylidene)pyrrolidin-2-one (Py23): White solid; yield: 46%; mp:
1
167-169 °C; H NMR (500 MHz, CDCl ) δ (ppm): 7.44 (d, J = 8.8 Hz, 2H), 7.31 (t, J = 2.7 Hz,
3
H
1H), 6.76 (d, J = 8.7 Hz, 2H), 6.69 (br, 1H), 3.57 (t, J = 6.6 Hz, 2H), 3.16 (td, J = 6.7, 2.4 Hz, 2H),
1
3
3.04 (s, 6H); C NMR (125 MHz, CDCl ) δ (ppm): 173.4, 150.4, 131.1, 130.8, 124.7, 123.8, 111.9,
3
C
+
40.1, 39.6, 26.4; HR-MS (ESI): m/z calcd for C H N ONa ([M+Na] ) 239.1155, found 239.1156.
1
3
16
2
2
8