New lead discovery of herbicide safener for metolachlor based on a scaffold-hopping strategy

Article abstract of DOI:10.3390/molecules25214986

The use of herbicide safeners can significantly alleviate herbicide injury to protect crop plants and expand the application scope of the existing herbicides in the field. Sanshools, which are well known as spices, are N-alkyl substituted compounds extracted from the Zanthoxylum species and have several essential physiological and pharmacological functions. Sanshools display excellent safener activity for the herbicide metolachlor in rice seedlings. However, the high cost of sanshools extraction and difficulties in the synthesis of their complicated chemical structures limit their utilization in agricultural fields. Thus, the present study designed and synthesized various N-alkyl amide derivatives via the scaffold-hopping strategy to solve the challenge of complicated structures and find novel potential safeners for the herbicide metolachlor. In total, 33 N-alkyl amide derivatives (2a–k, 3a–k, and 4a–k) were synthesized using amines and saturated and unsaturated fatty acids as starting materials through acylation and condensation. The identity of all the target compounds was well confirmed by¹H-NMR,¹³C-NMR, and high-resolution mass spectrometry (HRMS). The primary evaluation of safener activities for the compounds by the agar method indicated that most of the target compounds could protect rice seedlings from injury caused by metolachlor. Notably, compounds 2k and 4k displayed excellent herbicide safener activities on plant height and demonstrated relatively similar activities to the commercialized compound dichlormid. Moreover, we showed that compounds 2k and 4k had higher glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO) activities in rice seedlings, compared to the metolachlor treatment. In particular, 2k and 4k are safer for aquatic organisms than dichlormid. Results from the current work exhibit that compounds 2k and 4k have excellent crop safener activities toward rice and can, thus, be promising candidates for further structural optimization in rice protection.

Full text of DOI:10.3390/molecules25214986

molecules
Article
New Lead Discovery of Herbicide Safener for
Metolachlor Based on a Scaffold-Hopping Strategy
Xile Deng , Wenna Zheng, Qingcai Zhan, Yanan Deng, Yong Zhou and Lianyang Bai *
Hunan Provincial Key Laboratory for Biology and Control of Weeds, Biotechnology Research Institute,
Hunan Academy of Agricultural Sciences, Changsha 410125, China; dengxile@hunaas.cn (X.D.);
zhengwenna1994@163.com (W.Z.); zhanqc@hotmail.com (Q.Z.); dengyanan007@hunaas.cn (Y.D.);
qimiaobuchugan@126.com (Y.Z.)
*
Correspondence: lybai@hunaas.cn; Tel.: +86-0731-8469-2799
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 5 September 2020; Accepted: 26 October 2020; Published: 28 October 2020
Abstract: The use of herbicide safeners can significantly alleviate herbicide injury to protect crop
plants and expand the application scope of the existing herbicides in the field. Sanshools, which are
well known as spices, are N-alkyl substituted compounds extracted from the Zanthoxylum species
and have several essential physiological and pharmacological functions. Sanshools display excellent
safener activity for the herbicide metolachlor in rice seedlings. However, the high cost of sanshools
extraction and difficulties in the synthesis of their complicated chemical structures limit their utilization
in agricultural fields. Thus, the present study designed and synthesized various N-alkyl amide
derivatives via the scaffold-hopping strategy to solve the challenge of complicated structures and
find novel potential safeners for the herbicide metolachlor. In total, 33 N-alkyl amide derivatives
(2a–k, 3a–k, and 4a–k) were synthesized using amines and saturated and unsaturated fatty acids as
starting materials through acylation and condensation. The identity of all the target compounds was
1
13
well confirmed by H-NMR, C-NMR, and high-resolution mass spectrometry (HRMS). The primary
evaluation of safener activities for the compounds by the agar method indicated that most of the target
compounds could protect rice seedlings from injury caused by metolachlor. Notably, compounds 2k
and 4k displayed excellent herbicide safener activities on plant height and demonstrated relatively
similar activities to the commercialized compound dichlormid. Moreover, we showed that compounds
2k and 4k had higher glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT),
peroxidase (POD), and polyphenol oxidase (PPO) activities in rice seedlings, compared to the
metolachlor treatment. In particular, 2k and 4k are safer for aquatic organisms than dichlormid.
Results from the current work exhibit that compounds 2k and 4k have excellent crop safener
activities toward rice and can, thus, be promising candidates for further structural optimization in
rice protection.
Keywords: sanshools; synthesis; N-alkylamides; natural products; herbicide safener
1
. Introduction
Weeds compete with crop plants for various resources, such as nutrients, water, and sunlight,
which significantly affect crop productivity. Besides, weeds also increase crop protection costs because
they harbor pests and bacterial diseases [ ]. Herbicide application has been a prominent weed-control
measure in recent decades under field conditions [ ]. For example, metolachlor, one of the most
widely used chloroacetanilide herbicides, is applied to crops (e.g., rice, corn, and soybean) to control
pre-emergent and early post-emergent broadleaf and grass weeds [ 11]. However, the use of herbicides
1–3
4,5
6–
as metolachlor for weed controls is phytotoxic to crop plants [12–14].
Molecules 2020, 25, 4986; doi:10.3390/molecules25214986
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4986
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Herbicide phytotoxicity is mainly solved through several means: (i) developing selective herbicides
that are relatively safe to the crops; (ii) cultivating herbicide-resistance crop plants; (iii) using herbicide
safeners [15
most cost-effective and widely used method [18
–
17]. Among all these methods, the application of herbicide safeners is considered the
21]. Herbicide safeners are chemicals that reduce the
–
phytotoxicity of herbicides to crop plants without affecting the weed-control efficacy [21]. The first
commercial herbicide safener, 1,8-naphthalic anhydride (NA), was discovered by Gulf Oil Company in
1970 and can efficiently reduce the injury caused by thiocarbamate herbicides on corn [22]. Since then,
nearly twenty commercial safeners have been launched on the market, such as dichlormid, benoxacor,
fenclorim, cloquintocet-mexyl, and flurozle. These safeners are used to protect major cash crops
against pre-plant-incorporated or pre-emergence-applied herbicides, such as chloroacetanilides and
thiocarbamates [23,24].
The primary mechanism for safener action is the enhancement of herbicide detoxification in
safened plants [21]. The ability of safeners to enhance glutathione S-transferase (GST) activity has
been particularly well described in rice, maize, and wheat, among other crops [25–27]. In these
cases, safener treatments may induce transcriptional activation of specific GST genes and enhance the
glutathione S-transferase (GST) expression of the respective enzymes that catalyze the conjugation of
herbicides with glutathione (GSH) in crop plants to detoxify them [28]. Besides, detoxifying enzymes,
including superoxide dismutase (SOD) and peroxidase (POD), are also involved in the detoxification
of some herbicides [18,29].
In the application of herbicide safeners in nearly 40 years, it is noted that herbicide has shown
its biological activity to protect crops plants as “inert ingredients”, suggesting that they do not act
on the weeds directly, and their environmental toxicity environment is not confirmed [30]. However,
commercial safeners inevitably pose potential environmental pollution risks following widespread
and long-term use. For instance, it has been shown that some commercial herbicide safeners are toxic
to aquatic organisms, such as fish [31]. Meanwhile, the extensive application of herbicides alongside
herbicides has caused the emergence of weed resistance, which could increase phytotoxicity to crop
plants [9]. This fact suggests that the practice can lead to major practical problems in agricultural
production, such as dramatic crop productivity losses. Therefore, the identification and development
of novel eco-friendly safeners with high-efficacy is of great significance and urgency.
Sanshools are N-alkyl substituted compounds extracted from the Zanthoxylum species.
Sanshools are well-known as spices and have several essential physiological and pharmacological
functions. Sanshools and hydroxyl-sanshools are TRPV1 (a thermosensitive transient receptor potential
ion channel vanilloid) agonists in sensory neurons, exhibiting pungent quality [32
shows an inhibition effect on an NF1- and p53-deficient mouse glioma cell line, indicating their
potential antitumor activity [34]. Especially, hydroxy- -sanshool can enhance the activity of the
,33]. TRPV1 also
α
cholinergic system and increase the CREB/BDNF signaling pathway to attenuate scopolamine-induced
cognitive impairments [35]. Besides, hydroxy-α-sanshool may also block KCNK9 channels to induce
colonic motor activity in the rat proximal colon [36]. Naturally occurring products provide an
excellent basis for the discovery of novel pesticides, including insecticides, fungicides, herbicides,
and even herbicide safeners [18
of hydroxy- -sanshool,
and dihydroxy- -sanshool (Figure 1) was extracted from Szechuan pepper fruits by the supercritical
,25
,37
,38]. In our previous study, a mixture of sanshools consisting
α
/β/
ε
α
/β/ε
-sanshool,
γ
-sanshool, hydroxyl- -sanshool, dehydro- -sanshool,
γ
γ
α/β
fluid extraction (SFE) method. Interestingly, the mixture demonstrated good herbicide safener activity
for metolachlor in rice seedlings. However, the use of SFE to extract large quantities of sanshools is not
cost-effective. Meanwhile, the reported procedures for organic synthesis of some of these sanshools are
relatively complicated, mainly because of the construction of unsaturated long alkyl carbon chains with
specific double-bond configurations in the carbon terminal of the amide bonds [39–43]. These factors
have further limited the application of sanshools as herbicide safeners, indicating that their structures
need modification (including simplification of the structures).

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Figure 1. Chemical structures of sanshools extracted from Szechuan pepper fruits.
Scaffold hopping is a useful tool for screening new pesticides used in pharmaceutical chemistry [44].
Among all scaffold hopping approaches, chain shortening is feasible for obtaining derivatives/analogs
with improved potency by shortening or elongating flexible aliphatic chains (usually between two rings
in the pesticide molecules). It is supposed that hydroxy-
α/β/ε-sanshool, α/β/ε-sanshool, γ-sanshool,
hydroxyl- -sanshool, and dehydro- -sanshool could be considered as the main compounds to design
γ
γ
a series of sanshool derivatives (N-alkyl substituted analogs). The objective of the study was to
simplify the structures of sanshools by replacing the unsaturated long alkyl carbon chains with
saturated and unsaturated alkyl carbon chains with one to six carbon atoms, using the chain shortening
method (Scheme 1). These compounds were synthesized via the one-spot method, and their structures
1
were characterized by proton nuclear magnetic resonance ( H NMR), carbon 13 nuclear magnetic
13
resonance ( C NMR), and high-performance liquid chromatography spectrum (HRMS). Subsequently,
we evaluated their safener activities that alleviate metolachlor injury on rice seedlings, the action
mechanism, and acute toxicity (96h) in zebrafish (Danio rerio) embryos, using dichlormid, an amide-type
commercial herbicide safener, as the control. The results of this research could guide the design of new
herbicide safeners with high efficacy from natural products and lay the foundation for developing
safeners that are eco-friendly to aquatic organisms in rice fields.
Scheme 1. The design strategy of target compounds 2a–k, 3a–k, and 4a–k.

Molecules 2020, 25, 4986
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2
. Results and Discussion
2
.1. Synthesis and Characterization of Target Compounds 2a–k, 3a–k, and 4a–k
The one-spot condensation reaction was employed to obtain 43–89% yields of the target
compounds 2a 3a , and 4a , using substituted acids and amines as starting materials and
–
k,
–
k
–k
chlotripyrrolidinophosphonium hexafluophosphate (PyClOP) as a catalyst (Scheme 2) [45].
Scheme 2. The synthetic procedures of target compounds 1a–k, 2a–k, and 3a–k.
The structures of all the target compounds were well characterized by H NMR, 13C NMR,
1
1
and HRMS spectroscopies. The H NMR spectrum of all the target compounds exhibited the hydrogen
protons of amide bonds as singlets at 5.72–7.64 ppm and protons of hydroxyl moieties of 3a–k as
singlets at 2.59–4.44 ppm. Besides, the 13C NMR spectrum of all the target compounds showed the
resonance of amide bonds (carbonyl moiety) at 162.2–173.8 ppm. What’s more, HRMS data of all the
target compounds concurred with the calculated data based on chemical formula.
2
.2. The Phytotoxicity of Target Compounds 2a–k, 3a–k, 4a–k, and Dichlormid on Rice Seedlings
Results on the phytotoxicities of 8 mg/L of 2a 3a 4a k, and dichlormid on the growth of
–
k,
–
k,
–
rice seedlings (plant height, root length, and fresh weight) are shown in Table 1. The experimental
results on phytotoxicity were described as plant height, root length, and fresh weight. Compared to
non-treated controls, the growth indices of the target compounds and the positive control dichlormid
ranged from 81.7–99.4% for plant height, 78.2–99.2% for root length, and 84.0–97.4% for fresh weight.
The phytotoxicity testing results on rice plants revealed that the target compounds and dichlormid had
very low inhibitory effects on the growth of rice seedlings.

Molecules 2020, 25, 4986
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−
1
Table 1. Phytotoxicity of 7-day-old rice seedlings treated with 8 mg
·
L
compounds 2a–k, 3a–k,
4
a
–k
, and dichlormid (
D
). The plant height, root length, and fresh weight were measured as
phytotoxicity parameters.
Safening Effect (% of Non-Treated Control)
Compound
D
Plant Height
Root Length
Fresh Weight
93.1 ± 1.0
90.3 ± 0.5
95.9 ± 0.8
88.3 ± 0.7
95.8 ± 0.5
95.6 ± 0.2
96.9 ± 1.2
97.5 ± 0.6
95.2 ± 0.4
99.4 ± 0.3
97.0 ± 0.3
97.6 ± 0.8
98.3 ± 0.7
91.6 ± 0.8
98.9 ± 0.2
83.2 ± 0.5
92.8 ± 1.0
89.3 ± 0.6
98.9± 0.9
94.7 ± 0.8
81.7 ± 0.2
97.2 ± 0.4
91.7 ± 0.5
90.3 ± 0.1
90.6 ± 0.3
97.4 ± 0.8
82.4 ± 0.4
98.1 ± 1.1
90.5 ± 0.3
96.8 ± 0.5
95.9 ± 0.7
91.2 ± 0.3
98.5 ± 0.4
96.9 ± 0.2
89.5 ± 2.0
97.8 ± 0.5
90.7 ± 0.6
79.9 ± 0.4
81.1 ± 0.4
83.8 ± 0.2
81.9 ± 0.6
88.4 ± 0.2
86.3 ± 1.0
87.5 ± 2.4
84.6 ± 0.5
85.2 ± 0.8
92.8 ± 0.3
98.8 ± 0.2
78.2 ± 0.9
96.0 ± 0.5
86.5 ± 0.8
91.2 ± 0.7
98.5 ± 1.0
95.6 ± 0.4
91.5 ± 0.9
91.1 ± 0.6
87.1 ± 0.4
94.8 ± 0.7
90.0 ± 0.8
67.4 ± 0.9
81.7 ± 0.9
90.2 ± 1.0
92.4 ± 1.1
89.3 ± 0.8
97.4 ± 0.7
91.7 ± 0.2
87.5 ± 0.3
99.2 ± 0.7
93.4 ± 0.6
91.3 ± 0.5
95.1 ± 0.6
89.2 ± 0.8
88.7 ± 1.1
90.8 ± 0.7
92.9 ± 0.2
91.2 ± 0.6
84.0 ± 0.7
96.5 ± 0.3
84.4 ± 0.8
93.6 ± 1.1
97.4 ± 0.7
90.4 ± 0.2
96.5 ± 0.3
93.1 ± 0.6
87.4 ± 0.8
86.4 ± 0.6
96.7 ± 0.6
96.7 ± 1.1
84.2 ± 0.9
96.6 ± 0.3
88.5 ± 0.7
95.3 ± 1.1
91.2 ± 0.8
87.4 ± 1.1
87.4 ± 0.5
96.6 ± 0.2
95.0 ± 0.7
87.7 ± 0.7
93.5 ± 1.2
89.0 ± 0.7
87.8 ± 0.5
94.1 ± 0.4
2
a
2
b
2
c
2
d
2
e
2
f
2
g
2
h
2
2
i
j
2
k
3
a
3
b
3
c
3
d
3
e
3
f
3
g
3
h
3
3
i
j
3
k
4
a
4
b
4
c
4
d
4
e
4
f
4
g
4
h
4
4
i
j
4
k
The values represent the means of three replicates ± SE of each mean.
2
.3. Safening Effects and Structure-Activity Relationship of 2a–k, 3a–k, and 4a–k on Rice Seedlings
As shown in Table 2, the herbicide safener activities of target compounds 2a–k, 3a–k, and 4a–k
protected rice seedlings from metolachlor injury. Metolachlor (Mcl) significantly inhibited the growth
of 7-day-old rice seedlings; the plant height, root length, and fresh weight were inhibited by 43.6%,
56.1%, and 75.0%, respectively. Treatments with target compounds 2a–k, 3a–k, and 4a–k alleviated
rice seedlings injury caused by metolachlor to different extents, ranging from 42.8–84.3%, 56.4–83.0%,
and 76.8–87.9% for plant height, root length, and fresh weight, respectively. Unfortunately, the safener
activities of 2a–k, 3a–k, and 4a–k were less than those of dichlormid. However, the safener activities of
2
k
and 4k were very similar to that of dichlormid, with plant height relative values of 84.3% and 81.0%,
respectively. Further, the root length of compound 3h (83.0%) was very close to that of dichlormid.
On the other hand, 3h exhibited the best safener activity on fresh weight (87.9%).

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Table 2. Herbicide safening effect of 7-day-old rice seedlings treated with metolachlor. The combined
−
1
formulations were 8 mg
·
L
compounds 2a 3a
–
k
,
–k, and 4a–k/0.25 µM metolachlor (C + Mcl) and
−1
8
mg
·L
dichlormid/0.25
µM metolachlor (D + Mcl). The plant height, root length, and fresh weight
were measured as parameters.
Compared to the Blank Control (%)
Compound
Relative Plant Height
43.6 ± 0.4
Relative Root Length
Relative Fresh Weight
Mcl
D + Mcl
56.1 ± 2.0
83.2 ± 2.5
65.9 ± 2.0
59.8 ± 3.0
64.7 ± 1.5
67.1 ± 2.7
63.9 ± 2.1
74.6 ± 0.7
68.1 ± 1.7
73.4 ± 2.5
67.1 ± 2.7
71.0 ± 0.7
81.1 ± 1.1
64.4 ± 1.9
76.2 ± 0.6
64.7 ± 1.5
60.8 ± 4.4
65.8 ± 2.6
64.6 ± 3.0
71.0 ± 1.5
83.0 ± 3.9
60.2 ± 2.4
70.5 ± 1.1
59.4 ± 1.0
57.3 ± 3.1
66.5 ± 3.1
56.4 ± 1.0
63.6 ± 2.2
56.8 ± 1.3
67.4 ± 1.3
60.4 ± 1.2
64.8 ± 4.0
63.1 ± 1.2
60.5 ± 0.4
82.2 ± 1.0
75.0 ± 2.2
92.7 ± 1.3
85.6 ± 3.1
81.6 ± 1.0
79.6 ± 2.8
83.3 ± 3.3
82.7 ± 1.6
79.0 ± 0.2
83.1 ± 1.8
85.8 ± 2.3
83.3 ± 3.3
81.4 ± 0.7
81.3 ± 1.3
82.7 ± 2.1
85.3 ± 3.1
79.6 ± 2.8
85.2 ± 2.8
83.1 ± 2.3
79.8 ± 3.2
86.3 ± 2.4
87.9 ± 0.8
82.6 ± 2.6
84.3 ± 3.1
81.8 ± 2.9
79.5 ± 2.7
83.5 ± 2.3
82.2 ± 3.1
81.1 ± 2.6
78.9 ± 1.4
78.3 ± 1.7
77.5 ± 3.3
84.7 ± 3.5
84.8 ± 2.9
76.8 ± 1.9
84.9 ± 0.6
92.6 ± 0.9
53.8 ± 2.3
62.1 ± 2.3
56.3 ± 1.9
54.8 ± 2.8
52.7 ± 0.8
58.5 ± 2.9
62.2 ± 1.2
64.4 ± 2.2
54.8 ± 2.8
70.1 ± 1.7
84.3 ± 2.0
52.0 ± 0.9
50.0 ± 1.2
56.3 ± 1.9
50.1 ± 2.2
53.9 ± 0.9
47.2 ± 1.5
53.2 ± 0.7
43.2 ± 1.5
48.0 ± 0.9
50.5 ± 0.8
50.2 ± 1.7
45.0 ± 2.9
49.8 ± 1.2
44.3 ± 1.6
56.3 ± 2.3
42.8 ± 0.3
53.7 ± 1.6
49.8 ± 0.9
64.4 ± 1.7
49.2 ± 1.1
63.5 ± 0.4
81.0 ± 0.4
2
2
2
2
2
2
2
2
a + Mcl
b + Mcl
c + Mcl
d + Mcl
e + Mcl
f + Mcl
g + Mcl
h + Mcl
2
2
2
i + Mcl
j + Mcl
k + Mcl
3
3
3
3
3
3
3
3
a + Mcl
b + Mcl
c + Mcl
d + Mcl
e + Mcl
f + Mcl
g + Mcl
h + Mcl
3
3
3
i + Mcl
j + Mcl
k + Mcl
4
4
4
4
4
4
4
4
a + Mcl
b + Mcl
c + Mcl
d + Mcl
e + Mcl
f + Mcl
g + Mcl
h + Mcl
4
4
4
i + Mcl
j + Mcl
k + Mcl
The values present the means of three replicates ± SE of each mean.
A brief structure-activity relationship (SAR) of 2a
When the R groups of all the compounds were the same, most of the compounds 2a
isobutyl group were generally more active (plant height activity) than 3a with 2-hydroxy-2-methyl
propyl groups and 4a with 2-methyl allyl groups. In particular, with an increase in the length of
the carbon chain, the biological activity generally showed an upward trend compared to 2a and
, when R groups represent saturated carbon chains. 2k and 4k with the longest carbon chain
six carbons) substitution showed the best safener activity on plant height. On the contrary, the activity
of 3a on plant height was not affected by the length of the carbon chain. Similar to the activity on
–
k,
3a
–
k
, and 4a
–
k
was concluded as follows.
–
k
with N-substituted
–
k
–
k
–
k
4a–k
(
–k
plant length, compounds bearing the longest carbon chain (six carbons) moieties also exhibited the best
activity on root length, compared to 2a–k and 4a–k. However, 3b with (1E)-1-pentenyl (two carbons)
was more active compared to 3a–k. It should, however, be mentioned that when the number of carbon
atoms in the main carbon chain is the same as in the R group, the saturation of the carbon chain affects
the herbicide safener activity. On plant height, for instance, n-amyl substitution showed better or

Molecules 2020, 25, 4986
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similar activity, compared to the (1E)-1-pentenyl and (1E, 3E)-1,3-pentadienyl substitutions. However,
on root length and fresh weight, (1E)-1-pentenyl substitution showed less or very similar activity to
that of n-amyl and (1E, 3E)-1,3-pentadienyl substitutions.
It was apparent that the herbicide safener activity was influenced by various attributes of
the substitution groups, particularly the carbon length and degrees of saturation. Generally,
compounds bearing a long carbon length moiety showed higher safener activities. In all, among all the
target compounds, 2k and 4k, both with n-hexyl moieties and a different N-substitution (isobutyl moiety
and 2-methyl allyl moiety, respectively), showed the best activity on plant height and excellent activity
on root length and fresh weight. These two compounds (2k and 4k) could, therefore, be considered the
most potential candidates.
2
.4. Safening Effect of Compounds 2k, 4k, and Dichlormid in Rice at Lower Concentrations
To verify the safening effects of 2k and 4k, their herbicide safener activities were further tested
at lower concentrations. As shown in Figure 2, there was not an explicit relationship between the
concentrations and the safening effects of 2k 4k, and dichlormid on plant height, root length, and fresh
,
weight. Although dichlormid showed better activity than both 2k and 4k on both plant length and root
length at most concentrations, the activities of 2k and 4k were still substantial at some concentrations,
for example, 1 mg/L and 4 mg/L for plant height and 0.25 mg/L, 2 mg/L, and 4 mg/L for root length
(Figure 2a,b). For fresh weight, 2k was more active than 4k and dichlormid at 0.25 mg/L, 2 mg/L,
and 4 mg/L, while 4k showed the best safener activities at 0.5 mg/L and 1 mg/L (Figure 2c). These results
also indicated that 2k and 4k showed moderate to good safener activities, even at lower concentrations.
D+Mcl
2k+Mcl
k+Mcl
D+Mcl
1
00
2k+Mcl
k+Mcl
4
4
100
80
60
40
20
0
80
60
40
20
0
0.25
0.5
1
2
4
0.25
0.5
1
2
4
Concentrations (mg/L）
Concentrations (mg/L)
(
a)
(b)
D+Mcl
2k+Mcl
k+Mcl
100
4
8
6
4
2
0
0
0
0
0
0.25
0.5
1
2
4
Concentrations (mg/L)
(
c)
), and fresh weight (
4k, and dichlormid (
mg/L, 2 mg/L, and 4 mg/L, respectively. The values present the means of three replicates
each mean.
Figure 2. Plant height (
a
), root length (
b
c
) relative values of 0.25
) at 0.25 mg/L, 0.5 mg/L,
SE of
µM metolachlor
(
Mcl)-treated rice plants treated with compounds 2k
,
D
1
±

Molecules 2020, 25, 4986
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2
.5. Enzyme Activity Assay
GST can catalyze the conjugation of GSH and herbicide molecules in crop plants,
which subsequently improves the tolerance of crop plants to herbicides [46]. As shown in Figure 3a,
GST activity decreased significantly after the treatment of metolachlor on rice seedlings. However,
the results of the present study demonstrated that the GST activities of rice seedlings treated with
a combination of compounds 2k and 4k with metolachlor were greater than that of rice seedlings
treated with metolachlor alone and even higher than that of control check (CK). Except for dichlormid,
rice treated with compound 2k exhibited the best GST activity, which was almost 187% of the
metolachlor-treated group and 1.16 times of CK.
1
100
000
1
000
1
9
8
7
00
00
00
800
600
400
200
0
600
500
400
300
200
100
0
CK
Mcl
D+Mcl
2k+Mcl
4k+Mcl
CK
Mcl
D+Mcl
2k+Mcl
4k+Mcl
(
a)
(b)
4
50
00
50
00
50
00
50
00
3
2
000
500
4
3
3
2
2
1
1
2000
1
500
△
1000
5
00
0
50
0
CK
Mcl
D+Mcl
2k+Mcl
4k+Mcl
CK
Mcl
D+Mcl
2k+Mcl
4k+Mcl
(
c)
(d)
100
80
60
40
20
0
△
CK
Mcl
D+Mcl
2k+Mcl
4k+Mcl
(
e)
Figure 3. Compounds 2k, 4k, and dichlormid on enzyme activities of rice seedlings. (
a) GST,
(
b
) SOD, ( ) CAT, ( ) POD, ( ) PPO. The values present the means of three replicates SE of each
c
d
e
±
mean. GST, glutathione S-transferase; SOD, superoxide dismutase; CAT, catalase; POD, peroxidase;
PPO, polyphenol oxidases.

Molecules 2020, 25, 4986
9 of 18
To minimize toxicity caused by herbicides, crop plants have evolved their own protective
mechanisms, for instance, the antioxidant system that is comprised of a series of enzymes, such as
superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) [47–49]. These antioxidant enzyme
activities have also been reported to be associated with herbicide tolerance in crop plants [28]. When rice
seedlings were only treated by metolachlor, SOD, POD, and CAT activities decreased significantly,
compared to that of CK (Figure 3b–d). After the addition of compounds 2k and 4k, the SOD, POD,
and CAT activities increased dramatically. For 2k treatments, the SOD, POD, and CAT activities
increased by 208%, 120%, and 215%, respectively; for 4k treatments, the SOD, POD, and CAT activities
increased by 223%, 140%, and 233%, respectively. These results indicated that 2k and 4k could induce
antioxidant enzyme activities that were inhibited by metolachlor.
Polyphenol oxidases (PPOs) are peroxidases involved in the biosynthesis of black/brown pigment
and have played protective roles in crop plants against fungus, environmental stress, and herbicide
stress [50
which was extremely unfavorable for rice growth (Figure 3e). In rice seedlings treated with compounds
and 4k, the PPO activities increased by 147% and 155%, respectively, compared to metolachlor
,51]. The results showed that metolachlor inhibited the PPO activity of rice seedlings,
2
k
treatments. Thus, these target compounds 2k and 4k effectively alleviated the inhibition of PPO activity
by herbicide metolachlor.
2
.6. Acute Toxicity of Compounds 2k, 4k, and Dichlormid on Zebrafish Embryos
As dichlormid has been reported to pose potential risks to aquatic organisms, such as fish,
we further evaluated the acute toxicity (96 h) of 2k
frequently used to evaluate the toxicity of various compounds to aquatic organisms [52
,
4k, and dichlormid on zebrafish embryo; a model
53]. Dichlormid
,
displayed moderate acute toxicity (7.86 mg/L) to zebrafish embryos, while 2k and 4k displayed very
low acute toxicities, with LC50 (Lethal Concentration 50%) values of 38.29 mg/L and 39.28 mg/L,
respectively (Table 3). These results indicated that 2k and 4k were safer for aquatic organisms relative
to dichlormid. Besides, we would test the effects of 2k and 4k on other additional environmental
factors, such as soil factor, under real farm conditions in future work.
Table 3. Acute toxicities of compounds 2k, 4k, and dichlormid to zebrafish embryos at 96 h
after treatment.
Compound
LC50 (mg/L)
Regression Equation
R2
2
4
k
k
38.29
39.28
7.86
y = 1.7630 + 4.2719x
y = 1.7557 + 4.2376x
y = 1.9248 + 3.4345x
0.9451
0.9456
0.8849
dichlormid
3
. Materials and Methods
3
.1. Chemicals
Metolachlor (emulsifiable concentrate, 960 g/L), substituted fatty acids (purity 97–99%), isobutylamine
purity 99%), 2-methyl allylamine (purity 97%), 1-amino-2-methyl propan-2-ol (purity 98%),
chlorotripyrrolidinophosphoniumhexafluorophosphate(PyClOP,purity98%), andN,N-diisopropylethylamine
DIEA, 99%) were purchased from Jilin Chinese Academy of Sciences-Yanshen Technology Co., Ltd.
Jilin, Changchun, China). All solvents were bought from Sinopharm Chemical Reagent Co., Ltd. (Beijing,
China) and further distilled before use. Thin-layer chromatography (TLC) was performed with Silicagel GF254
Merck KGaA, Darmstadt, Germany). The isolation and purification were carried out via silica gel column
chromatography (200 mesh). Melting points (Mp) were measured by a Hanon MP100 automatic melting point
(
(
(
(
1
13
apparatus (Jinan Hanon Instruments Co., Ltd., Jinan, China) and autocorrected. H NMR and C NMR were
recorded on a Bruker Avance-300 spectrometer operating at 300 MHz ( H) and 75 MHz ( C), respectively,
using tetramethylsilane (TMS) as an internal standard (solvent CDCl or DMSO-d ). Chemical shifts ( ) were
1
13
δ
3
6

Molecules 2020, 25, 4986
10 of 18
reported in parts per million (ppm), and coupling constants (J) were in Hz. HRMS was analyzed on a Varian
.0 T FTICR-MS instrument (Varian IonSpec, Lake Forest, CA, USA).
7
3
.2. General Procedures for the Synthesis of N-Alkyl Amides 2a–k, 3a–k, and 4a–k
N-alkyl amides 2a 3a , and 4a were synthesized using the method reported in the previous
reference. The PyClOP (10 mmol) was added to a solution of carboxylic acid 1a (10 mmol),
–k
,
–k
–k
–
k
amine (butylamine, 2-methyl allylamine, or 1-amino-2-methyl propan-2-ol, 10 mmol), and DIEA
◦
(
20 mmol) in 30 mL of dimethylformamide (DMF) at 0 C. Next, the mixture solution was continuously
stirred for 12 h at room temperature and then diluted with 20 mL water and 20 mL ethyl acetate.
The organic layer was washed with 100 mL brine twice, 100 mL dilute HCl (1 mol/L) twice, 100 mL
water twice, 100 mL 5% NaHCO twice, and 100 mL brine twice. The resulting mixture solution was
3
concentrated under vacuum and further purified by flash chromatography on a silica gel column
(
ethyl acetate: petroleum ether, 1:6) to afford the N-alkyl amides 2a
–
k
,
3a
–
k, and 4a
–
k
(yields 34–84%)
1
13
as colorless oils, yellow oils, white solids, or yellow solids. H NMR and C NMR spectrums of target
compounds 2a–k, 3a–k, and 4a–k see the Supplementary Materials.
1
N-isobutylacetamide (2a). Yellow liquid; Yield 58%; H NMR (300 MHz, CDCl ):
δ 5.73 (s, 1H,
3
NH), 3.03–3.07 (m, 2H, CH ), 1.97 (s, 3H, CH ), 1.68–1.81 (m, 1H, CH), 0.89 (d, J = 6.0 Hz, 6H, CH );
2
3
3
1
3
+
C NMR (75 MHz, CDCl3):
16.1070; found, 116.1071.
N-isobutylacrylamide (2b). Yellow liquid; Yield 53%; H NMR (300 MHz, CDCl ):
δ
170.3, 46.9, 28.3, 23.1, 19.9, 19.7; HRMS calcd. for C H NO ([M + H] ),
6
13
1
1
δ
6.89 (s, 1H,
3
NH), 6.19 (d, J = 1.6 Hz, 1H, CH), 6.17 (s, 1H, CH), 5.52 (dd, J = 6.8, 5.2 Hz, 1H, CH), 3.05 (d, J = 13.0 Hz,
1
3
2
δ
H, CH ), 1.74 (dp, J = 13.4, 6.7 Hz, 1H, CH), 0.84 (d, J = 6.7 Hz, 6H, CH ); C NMR (75 MHz, CDCl3):
2 3
+
165.7, 131.0, 125.8, 46.9, 28.4, 20.0; HRMS calcd. for C H NO ([M + H] ), 128.1070; found, 128.1069.
7 13
1
N-isobutylpropionamide (2c). White liquid; Yield 34%; H NMR (300 MHz, CDCl ):
δ
6.04 (s, 1H,
3
NH), 3.00 (t, J = 6.4 Hz, 2H, CH ), 2.16 (q, J = 7.6 Hz, 2H, CH ), 1.71 (dq, J = 13.5, 6.7 Hz, 1H, CH),
2
2
13
1
2
.09 (t, J = 7.6 Hz, 3H, CH ), 0.84 (dd, J = 6.7 Hz, 6H, CH ); C NMR (75 MHz, CDCl3): δ 172.7, 45.9,
3 3
8.4, 28.0, 19.9, 9.9; HRMS calcd. for C H NO ([M + H] ), 130.1226; found, 130.1225.
+
7
15
◦
1
(
E)-N-isobutylbut-2-enamide (2d). White solid; Mp 67.5–69.5 C; Yield 51%; H NMR (300 MHz,
CDCl3): 6.83 (dq, J = 15.1, 6.9 Hz, 1H, CH), 5.79 (dq, J = 15.2, 1.7 Hz, 1H, CH), 5.48 (s, 1H, NH),
δ
3
0
.14 (dd, J = 6.8, 6.1 Hz, 2H, CH ), 1.84 (dd, J = 6.9, 1.7 Hz, 3H, CH ), 1.76 (dt, J = 13.4, 6.7 Hz, 1H, CH),
2
3
3
1
.91 (d, J = 6.7 Hz, 6H, CH3); C NMR (75 MHz, CDCl ):
δ
166.1, 139.2, 125.2, 46.7, 28.5, 20.0, 17.5;
3
+
HRMS calcd. for C H NO ([M + H] ), 142.1226; found, 142.1225.
8
15
1
N-isobutylbutyramide (2e). White liquid; Yield 57%; H NMR (300 MHz, CDCl ): δ 5.78 (s, 1H, NH),
3
3
0
.04–3.12 (m, 2H, CH ), 2.17 (dd, J = 8.2, 6.8 Hz, 2H, CH ), 1.61–1.81 (m, 3H, CH + CH ),
2
2
2
1
3
.88–0.98 (m, 9H, CH3); C NMR (75 MHz, CDCl3):
δ 171.7, 45.8, 37.3, 28.0, 19.9, 18.7, 13.4; HRMS calcd.
+
for C H NO ([M + H] ), 144.1383; found, 144.1381.
8
17
◦
1
(
E)-N-isobutylpent-2-enamide (2f). Yellow solid; Mp 41.8–42.5 C; Yield 72%; H NMR (300 MHz,
CDCl ): δ 6.87 (m, J = 15.3, 6.4 Hz, 1H, CH), 5.82 (m, J = 1.7 Hz, 1H, CH), 5.77 (t, J = 1.7 Hz, 1H, NH),
3
3
.14 (dd, J = 6.0, 6.1 Hz, 2H, CH ), 2.19 (ddd, J = 7.5, 6.4, 1.7 Hz, 2H, CH ), 1.72–1.90 (m, 1H, CH),
2
2
13
1.05 (t, J = 7.4 Hz, 3H, CH ), 0.92 (d, J = 6.7 Hz, 6H, CH ); C NMR (75 MHz, CDCl ):
δ
166.3, 145.6,
3
3
3
+
1
22.8, 46.8, 28.5, 25.0, 20.1, 12.4; HRMS calcd. for C H NO ([M + H] ), 156.1383; found, 156.1382.
9
17
1
N-isobutylpentanamide (2g). White liquid; Yield 80%; H NMR (300 MHz, CDCl ):
δ
5.73 (s, 1H,
3
NH), 3.05–3.10 (m, 2H, CH ), 2.16–2.21 (m, 2H, CH ), 1.72–1.81 (m, 1H, CH), 1.57–1.67 (m, 2H, CH ),
2
2
2
13
1
3
.35 (dd, J = 15.0, 7.4 Hz, 2H, CH ), 0.90–0.94 (m, 9H, CH ); C NMR (75 MHz, CDCl ): δ 171.9, 45.8,
2 3 3
5.0, 28.0, 27.5, 21.7, 20.0, 13.6; HRMS calcd. for C H NO ([M + H] ), 158.1539; found, 158.1538.
+
9
19
1
(
E)-N-isobutylhex-2-enamide (2h). White liquid; Yield 83%; H NMR (300 MHz, CDCl3):
δ
6.79 (dt, J = 15.2, 7.0 Hz, 1H, CH ), 5.94 (s, 1H, NH), 5.79 (dt, J = 15.3, 1.5 Hz, 1H, CH),
2
3
.11 (dd, J = 6.8, 6.2 Hz, 2H, CH ), 2.16–2.06 (m, 2H, CH ), 1.76 (dq, J = 13.4, 6.7 Hz, 1H, CH ),
2
2
2
13
1
.44 (h, J = 7.3 Hz, 2H, CH ), 0.95–0.80 (m, 9H, CH ); C NMR (75 MHz, CDCl ):
δ
166.2, 144.2, 123.8,
2
3
3
+
4
6.7, 34.0, 28.5, 21.4, 20.1, 13.6; HRMS calcd. for C H NO ([M + H] ), 170.1539; found, 170.1538.
10
19

Molecules 2020, 25, 4986
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◦
1
(
2E, 4E)-N-isobutylhexa-2, 4-dienamide (2i). White solid; Mp 106.5–107.0 C; Yield 81%; H NMR
(
300 MHz, CDCl3): 7.18 (dd, J = 14.9, 9.9 Hz, 1H, CH), 6.01–6.19 (m, 2H, CH), 5.73 (d, J = 15.0 Hz, 1H,
δ
CH), 5.46 (s, 1H, NH), 3.14–3.18 (m, 2H, CH ), 1.82–1.84 (m, 4H, CH + CH ), 0.92 (d, J = 6.7 Hz, 6H,
2
3
CH3); 13C NMR (75 MHz, CDCl ):
δ 166.5, 140.7, 137.2, 129.7, 121.9, 46.9, 28.5, 20.1, 18.4; HRMS calcd.
for C H NO ([M + H] ), 168.1383; found, 168.1381.
3
+
10
17
1
N-isobutylhexanamide (2j). White liquid; Yield 84%; H NMR (300 MHz, CDCl ):
δ
5.88 (s, 1H,
3
NH), 3.07 (dd, J = 6.9, 6.0 Hz, 2H, CH ), 2.16–2.21 (m, 2H, CH ), 1.78 (dq, J = 13.4, 6.7 Hz, 1H, CH),
2
2
13
1
δ
.62–1.69 (m, 2H, CH ), 1.26–1.35 (m, 4H, CH ), 0.86–0.96 (m, 9H, CH ); C NMR (75 MHz, CDCl ):
2 2 3 3
171.9, 45.8, 35.3, 30.8, 28.2, 28.0, 25.0, 21.8, 20.0, 13.8; HRMS calcd. for C H NO ([M + H] ), 172.1696;
10 21
+
found, 172.1694.
N-isobutylheptanamide (2k). White liquid; Yield 63%; 1H NMR (300 MHz, CDCl3):
5.83 (s, 1H, NH), 3.06 (t, J = 6.4 Hz, 2H, CH ), 2.13–2.22 (m, 2H, CH ), 1.76 (tq, J = 13.4, 6.7 Hz, 1H, CH),
δ
2
2
13
1
δ
.57–1.67 (m, 2H, CH ), 1.28–1.33 (m, 6H, CH ), 0.85–0.92 (m, 9H, CH ); C NMR (75 MHz, DMSO-d )
2 2 3 6
+
171.9, 45.8, 35.3, 31.0, 28.3, 28.0, 25.3, 21.9, 20.0, 13.8; HRMS calcd. for C H NO ([M + H] ), 186.1852;
11 23
found, 186.1842.
1
N-(2-hydroxy-2-methylpropyl)acetamide (3a). Colorless liquid; Yield 57%; H NMR (300 MHz,
CDCl ): 6.29 (s, 1H, NH), 3.25 (d, J = 6.0 Hz, 2H, CH ), 2.98 (s, 1H, OH), 2.03 (s, 3H, CH ), 1.22 (s, 6H,
δ
3
1
2
3
3
+
CH3); C NMR (75 MHz, CDCl3):
δ 171.1, 70.7, 50.4, 27.1, 23.0; HRMS calcd. for C H NO ([M + H] ),
6 13 2
1
32.1019; found, 132.1018.
N-(2-hydroxy-2-methylpropyl)acrylamide (3b). Colorless liquid; Mp 67.5–68.2 C; Yield 54%;
H NMR (300 MHz, CDCl3): 6.50 (s, 1H, NH), 6.12–6.33 (m, 2H, CH ), 5.66 (dd, J = 9.9, 1.8 Hz, 1H,
◦
1
δ
2
13
CH), 3.34 (d, J = 6.1 Hz, 2H, CH ), 3.09 (s, 1H, OH), 1.23 (s, 6H, CH ); C NMR (75 MHz, CDCl3):
δ
2
3
+
166.6, 130.6, 126.7, 70.8, 50.4, 27.2; HRMS calcd. for C H NO ([M + H] ), 144.1019; found, 144.1018.
7 13 2
1
N-(2-hydroxy-2-methylpropyl)propionamide (3c). Colorless liquid; Yield 61%; H NMR (300
MHz, CDCl3): 7.61 (s, 1H, NH), 4.44 (s, 1H, OH), 3.00 (d, J = 6.1 Hz, 2H, CH ), 2.11 (q, J = 7.6 Hz, 2H,
CH ), 1.02 (s, 6H, CH ), 0.98 (t, J = 7.6 Hz, 3H, CH ); C NMR (75 MHz, CDCl3):
δ
2
1
3
δ
173.8, 70.0, 50.1,
2
3
3
+
2
9.0, 27.7, 10.7; HRMS calcd. for C H NO ([M + H] ), 146.1176; found, 146.1173.
7 15 2
1
(
E)-N-(2-hydroxy-2-methylpropyl)but-2-enamide (3d). Colorless liquid; Yield 47%; H NMR
(300 MHz, DMSO-d6):
δ 6.86 (dq, J = 15.2, 6.9 Hz, 1H, CH), 5.94 (s, 1H, NH), 5.81–5.88 (m, 1H, CH),
3
.32 (d, J = 6.1 Hz, 2H, CH ), 2.59 (s, 1H, OH), 1.86 (dd, J = 6.9, 1.7 Hz, 3H, CH ), 1.23 (s, 6H, CH );
2
3
3
13
C NMR (75 MHz, CDCl ):
δ 167.1, 140.2, 124.8, 70.8, 50.4, 27.1, 17.6; HRMS calcd. for C H NO
8 15 2
3
+
([M + H] ), 158.1176; found, 158.1175.
◦
1
N-(2-hydroxy-2-methylpropyl)butyramide (3e). White solid; Mp 38.9–39.2 C; Yield 56%; H NMR
300 MHz, DMSO-d6): 7.64 (s, 1H, NH), 4.44 (s, 1H, OH), 3.01 (d, J = 6.1 Hz, 2H, CH ), 2.08 (t, J = 6.0 Hz,
H, CH ), 1.50 (h, J = 7.3 Hz, 2H, CH ), 1.03 (s, 6H, CH ), 0.84 (t, J = 7.4 Hz, 3H, CH ); C NMR
(
2
δ
2
13
2
2
3
3
+
(
1
75 MHz, CDCl3):
60.1332; found, 160.1331.
E)-N-(2-hydroxy-2-methylpropyl)pent-2-enamide (3f). Yellow liquid; Yield 61%; H NMR
300 MHz, CDCl3): 6.88–6.93 (m, 1H, CH), 6.09 (s, 1H, NH), 5.81 (d, J = 15.3 Hz, 1H, CH),
δ 172.9, 100.0, 70.0, 50.1, 27.7, 19.3, 14.2; HRMS calcd. for C H NO ([M + H] ),
8 17 2
1
(
(
δ
3
3
.32 (d, J = 6.1 Hz, 2H, CH ), 2.85 (s, 1H, OH), 2.18–2.23 (m, 2H, CH ), 1.22 (s, 6H, CH ), 1.05 (t, J = 7.4 Hz,
2
2
3
H, CH3); 1 C NMR (75 MHz, CDCl ):
3
δ
162.2, 141.3, 117.2, 65.5, 45.3, 21.9, 19.8, 7.1; HRMS calcd. for
3
+
C H NO ([M + H] ), 172.1332; found, 172.1330.
9
17
2
◦
N-(2-hydroxy-2-methylpropyl)pentanamide (3g). White solid; Mp 64.2–65.2 C; Yield 43%;
H NMR (300 MHz, DMSO-d6): 7.64 (s, 1H, NH), 4.43 (s, 1H, OH), 3.01 (d, J = 6.0 Hz, 2H, CH2),
.11 (t, J = 7.4 Hz, 2H, CH ), 1.47 (p, J = 7.4 Hz, 2H, CH ), 1.22–1.29 (m, 2H, CH ), 1.02 (s, 6H, CH ),
1
δ
2
0
2
2
2
3
13
.85 (t, J = 7.4 Hz, 3H, CH3); C NMR (75 MHz, CDCl ):
δ 173.0, 70.0, 50.1, 35.6, 28.1, 27.7, 22.3, 14.3;
3
+
HRMS calcd. for C H NO ([M + H] ), 174.1489; found, 174.1487.
9
19
2
◦
(
E)-N-(2-hydroxy-2-methylpropyl)hex-2-enamide (3h). White solid; Mp 41.3–42.0 C; Yield 43%;
H NMR (300 MHz, CDCl3): 6.79–6.89 (dt, J = 15.3, 7.0 Hz, 1H, CH), 6.41 (s, 1H, NH), 5.82–5.88
d, J = 15.3 Hz, 1H, CH), 3.33 (s, 2H, CH ), 3.30 (s, 1H, OH), 2.15 (q, J = 7.3 Hz, 2H, CH ), 1.40–1.52
1
δ
(
2
2

Molecules 2020, 25, 4986
12 of 18
1
3
(
1
m, 2H, CH ), 1.22 (s, 6H, CH ), 0.90 (t, J = 7.4 Hz, 3H, CH ); C NMR (75 MHz, CDCl ):
δ
167.2,
2
3
3
3
+
45.0, 123.3, 70.8, 50.4, 34.0, 27.1, 21.4, 13.6; HRMS calcd. for C H NO ([M + H] ), 186.1489; found,
10 19 2
1
86.1488.
◦
(
2E,4E)-N-(2-hydroxy-2-methylpropyl)hexa-2,4-dienamide (3i). White solid; Mp 142.2–142.7 C;
1
Yield 41%; H NMR (300 MHz, CDCl ):
δ 7.20 (dd, J = 15.0, 9.8 Hz, 1H, CH), 6.03–6.20 (m, 3H, CH+NH),
3
5
.77 (d, J = 15.0 Hz, 1H, CH), 3.34 (d, J = 6.1 Hz, 2H, CH ), 2.82 (s, 1H, OH), 1.83 (d, J = 5.7 Hz, 3H,
2
13
CH ), 1.23 (s, 6H, CH ); C NMR (75 MHz, CDCl ):
δ
167.6, 141.5, 138.0, 129.6, 121.2, 70.8, 50.5, 27.1,
3
3
3
+
1
8.5; HRMS calcd. for C H NO ([M + H] ), 184.1332; found, 184.1331.
10 17 2
◦
1
N-(2-hydroxy-2-methylpropyl)hexanamide (3j). White solid; Mp 53.8–54.3 C; Yield 52%; H NMR
300 MHz, DMSO-d6): 7.64 (m, 1H, NH), 4.43 (s, 1H, OH), 3.00 (d, J = 6.1 Hz, 2H, CH ), 2.10 (t, J = 7.4 Hz,
(
δ
2
2
H, CH ), 1.49 (p, J = 7.4 Hz, 2H, CH ), 1.18–1.29 (m, 4H, CH ), 1.02 (s, 6H, CH ), 0.85 (t, J = 7.1 Hz, 3H,
2
3
2
2
3
1
CH3); C NMR (75 MHz, DMSO-d ):
for C H NO ([M + H] ), 188.1645; found, 188.1643.
N-(2-hydroxy-2-methylpropyl)heptanamide (3k). White liquid; Yield 43%; H NMR (300 MHz,
DMSO-d6): 7.64 (s, 1H, NH), 4.43 (s, 1H, OH), 3.37 (s, 2H, CH ), 3.00 (d, J = 6.1 Hz, 2H, CH ),
δ 173.0, 70.0, 50.1, 35.8, 31.4, 27.7, 25.6, 22.4, 14.4; HRMS calcd.
6
+
10
21
2
1
δ
2
2
2
.10 (t, J = 6.0 Hz, 2H, CH ), 1.49 (p, J = 7.4 Hz, 2H, CH ), 1.24 (dqd, J = 22.7, 7.9, 7.4, 2.0 Hz, 4H, CH ),
2
2
2
13
1.02 (s, 6H, CH ), 0.84 (t, J = 7.1 Hz, 3H, CH ); C NMR (75 MHz, DMSO-d ):
δ
173.0, 70.0, 50.1, 35.8,
3
3
6
+
3
1.4, 27.7, 25.6, 22.4, 14.4; HRMS calcd. for C H NO ([M + H] ), 202.1802; found, 202.1800.
N-(2-methylallyl)acetamide (4a). Yellow liquid; Yield 56%; H NMR (300 MHz, CDCl3):
5.80 (s, 1H, NH), 4.82–4.84 (s, 2H, CH ), 3.80 (d, J = 6.0 Hz, 2H, CH ), 2.02 (s, 3H, CH ), 1.73 (m, 3H, CH );
1
1
23
2
1
δ
2
2
3
3
+
1
3
C NMR (75 MHz, CDCl3):
14.0913; found, 114.0915.
δ
170.2, 141.7, 110.4, 44.8, 22.8, 20.1; HRMS calcd. for C H NO ([M+H] ),
6
11
1
1
N-(2-methylallyl)acrylamide (4b). White liquid; Yield 64%; H NMR (300 MHz, CDCl ):
δ
6.31
3
(
1
(
dd, J = 17.0, 1.7 Hz, 1H, CH), 6.15 (dd, J = 17.0, 10.1 Hz, 1H, CH ), 5.94 (s, 1H, NH), 5.66 (dd, J = 10.0,
2
13
.7 Hz, 1H, CH), 4.85 (h, J = 1.1 Hz, 2H, CH ), 3.89 (d, J = 6.1 Hz, 2H, CH ), 1.75 (s, 3H, CH ); C NMR
2 2 3
+
75 MHz, CDCl3):
δ
165.8, 141.6, 130.9, 126.1, 110.9, 45.0, 20.3; HRMS calcd. for C H NO ([M + H] ),
7 11
1
26.0913; found, 126.0912.
1
N-(2-methylallyl)propionamide (4c). Yellow liquid; Yield 69%; H NMR (300 MHz, CDCl ):
3
δ
5.57 (s, 1H, NH), 4.81–4.83 (s. 2H, CH), 3.91 (d, J = 6.0 Hz, 2H, CH ), 2.24 (q, J = 7.6 Hz, 2H, CH ),
2
2
13
1.73 (s, 3H, CH ), 1.18 (t, J = 7.6 Hz, 3H, CH ); C NMR (75 MHz, CDCl ):
δ
165.8, 141.6, 130.9, 126.1,
3
3
3
+
1
10.9, 45.0, 20.2; HRMS calcd. for C H NO ([M + H] ), 128.1070; found, 128.1070.
7
13
1
(
E)-N-(2-methylallyl)but-2-enamide (4d). Yellow liquid; Yield 59%; H NMR (300 MHz, CDCl ):
3
δ
6.87 (dq, J = 15.1, 6.9 Hz, 1H, CH), 5.84 (dq, J = 15.2, 1.7 Hz, 1H, CH), 5.62 (s, 1H, NH), 4.84 (p, J = 1.2 Hz,
H, CH ), 3.88 (d, J = 6.1 Hz, 2H, CH ), 1.87 (dd, J = 6.9, 1.7 Hz, 3H, CH ), 1.75 (d, J = 1.1 Hz, 3H,
2
2
3
2
3
1
CH3); C NMR (75 MHz, CDCl ):
C H NO ([M + H] ), 140.1070; found, 140.1068.
δ 166.0, 141.9, 139.7, 124.9, 110.7, 44.8, 20.2, 17.6; HRMS calcd. for
3
+
8
13
1
N-(2-methylallyl)butyramide (4e). Colorless liquid; Yield 54%; H NMR (300 MHz, CDCl ):
3
δ
6.85–6.93 (m, 1H, CH), 5.79 (dt, J = 15.3, 1.7 Hz, 1H, CH), 5.62 (s, 1H, NH), 4.83 (p, J = 1.1 Hz, 2H,
CH ), 3.88 (d, J = 6.1 Hz, 2H, CH ), 2.21 (ddd, J = 7.7, 6.4, 1.5 Hz, 2H, CH ), 1.74 (s, 3H, CH ), 1.06 (s, 3H,
2
2
2
3
13
CH3); C NMR (75 MHz, CDCl ):
C H NO ([M + H] ), 142.1226; found, 142.1225.
δ 172.9, 141.9, 110.4, 44.7, 38.3, 20.2, 19.1, 13.6; HRMS calcd. for
3
+
8
15
1
(
E)-N-(2-methylallyl)pent-2-enamide (4f). Colorless liquid; Yield 53%; H NMR (300 MHz, CDCl ):
3
δ
6.85–69.5 (m, 1H, CH), 5.62–5.82 (dt, J = 15.3, 1.7 Hz, 1H, CH), 5.62 (s, 1H, NH), 4.83 (q, J = 1.1 Hz,
H, CH ), 3.88 (d, J = 6.1 Hz, 2H, CH ), 2.21 (ddd, J = 7.7, 6.4, 1.5 Hz, 2H, CH ), 1.04 (s, 3H, CH ),
2
2
2
2
3
13
1
.06 (t, J = 7.4 Hz, 3H, CH3); C NMR (75 MHz, CDCl ):
δ
166.2, 146.0, 141.9, 122.6, 110.7, 44.9, 25.0,
3
+
2
0.3, 12.4; HRMS calcd. for C H NO ([M + H] ), 154.1226; found, 154.1224.
9
15
1
N-(2-methylallyl)pentanamide (4g). Yellow liquid; Yield 61%; H NMR (300 MHz, CDCl ):
3
δ
5.85 (s, 1H, NH), 4.92–4.78 (m, 2H, CH ), 3.81 (d, J = 6.0 Hz, 2H, CH ), 2.19–2.28 (m, 2H, CH ),
2
2
2
1
.74 (d, J = 2.9 Hz, 3H, CH ), 1.57–1.70 (m, 2H, CH ), 1.30–1.44 (m, 2H, CH ), 0.93 (t, J = 7.3 Hz, 3H,
3 2 2

Molecules 2020, 25, 4986
13 of 18
CH ); 13C NMR (75 MHz, CDCl ):
δ
173.2, 141.9, 110.3, 44.6, 36.2, 27.8, 22.2, 20.1, 13.6; HRMS calcd. for
3
3
+
C H NO ([M + H] ), 156.1383; found, 156.1381.
9
17
◦
(
E)-N-(2-methylallyl)hex-2-enamide (4h).
White solid; Mp 37.0–38.8 C; Yield 57%;
1
H NMR (300 MHz, CDCl3):
δ
6.85 (dt, 1H, J = 15.3, 7.0 Hz, CH), 5.80 (dt, 1H, J = 15.3, 1.5 Hz,
CH), 5.66 (s, 1H, NH), 4.83 (q, J = 1.2 Hz, 2H, CH ), 3.87 (d, J = 6.1 Hz, 2H, CH ), 2.15 (qd, J = 7.1,
2
2
1
.5 Hz, 2H, CH ), 1.73–1.75 (m, 3H, CH ), 1.47 (h, J = 7.3 Hz, 2H, CH ), 0.92 (t, J = 7.4 Hz, 3H, CH );
2 3 2 3
13
C NMR (75 MHz, CDCl ):
δ 166.0, 144.7, 142.0, 123.5, 110.8, 44.9, 34.0, 21.4, 20.3, 13.6; HRMS calcd.
3
+
for C H NO ([M + H] ), 168.1383; found, 168.1381.
10
17
◦
(
2E, 4E)-N-(2-methylallyl)hexa-2,4-dienamide (4i). Yellow solid; Mp 66.3–67.1 C; Yield 49%;
1
H NMR (300 MHz, CDCl3): 7.17–7.25 (m, 1H, CH), 6.03–6.21 (m, 2H, CH), 5.77 (d, J = 14.9 Hz, 1H,
δ
CH), 5.58 (s, 1H, NH), 4.84 (q, J = 1.2 Hz, 2H, CH ), 3.89 (d, J = 6.1 Hz, 2H, CH ), 1.83 (d, J = 5.8 Hz, 3H,
2
2
13
CH ), 1.75 (s, 3H, CH ); C NMR (75 MHz, CDCl ):
δ
166.5, 141.9, 141.2, 137.6, 129.6, 121.4, 110.8, 45.0,
3
3
3
+
2
0.3, 18.4; HRMS calcd. for C H NO ([M + H] ), 166.1226; found, 166.1225.
10 15
1
N-(2-methylallyl)hexanamide (4j). Yellow liquid; Yield 61%; H NMR (300 MHz, CDCl ):
3
δ
2
0
5.72 (s, 1H, NH), 4.84 (dt, J = 2.6, 1.3 Hz, 2H, CH ), 3.82 (dd, J = 6.0, 1.4 Hz, 2H, CH ),
2
2
.20–2.25 (m, 2H, CH ), 1.74–1.75 (m, 3H, CH ), 1.62–1.69 (m, 2H, CH ), 1.29–1.38 (m, 4H, CH ),
2
3
2
2
1
3
.89–0.93 (m, 3H, CH3); C NMR (75 MHz, CDCl ):
δ 173.1, 141.9, 110.4, 44.7, 36.4, 31.3, 25.4, 22.2, 20.1,
3
+
1
3.7; HRMS calcd. for C H NO ([M + H] ), 170.1539; found, 170.1538.
1
0
19
1
N-(2-methylallyl)heptanamide (4k). Colorless liquid; Yield 67%; H NMR (300 MHz, CDCl ):
3
δ
5.87 (s, 1H, NH), 4.82 (p, J = 1.4 Hz, 2H, CH ), 3.81 (d, J = 6.0 Hz, 2H, CH ), 2.19–2.24 (m, 2H, CH ),
2
2
2
13
1
.73 (s, 3H, CH ), 1.62–1.67 (m, 2H, CH ), 1.26–1.35 (m, 6H, CH ), 0.86–0.90 (m, 3H, CH ); C NMR
3 2 2 3
(
75 MHz, CDCl3):
δ 173.2, 141.9, 110.3, 77.4, 77.0, 76.6, 44.6, 36.4, 31.4, 28.8, 25.7, 22.3, 20.1, 13.8;
+
HRMS calcd. for C H NO ([M + H] ), 184.1696; found, 184.1694.
1
1
21
3
.3. Phytotoxicity and Safening Effects of Compounds 2a–k, 3a–k, and 4a–k on Rice Seedlings
Full rice seeds (Oryza sativa) of uniform sizes were germinated via the reported method [18].
Briefly, the seeds were sterilized by 5% sodium hypochlorite solution and then washed in deionized
water. Then, sterile seeds were soaked in deionized water for 24 h and germinated for another 36 h in a
◦
plant growth cabinet (28 ± 1 C) in the dark.
The phytotoxicity and safener activities of target compounds 2a–k, 3a–k, and 4a–k on rice
seedlings were evaluated under greenhouse conditions according to the agar culture method we
◦
reported previously [54
,
55]. First, a 12 g agar strip was added to 3 L deionized water at 100 C and
constantly stirred over heat until the complete dissolution of the agar strip. Second, the mixture was
diluted to 4 L with deionized water to obtain a 0.3% agar medium solution. Then, a standard working
solution of metolachlor (Mcl, 960 g/L) was obtained by diluting 0.1 mL emulsifiable concentrate
−
1
◦
(
960 g L ) in 99.9 mL deionized water. A 600 mL aliquot of the agar medium was cooled to 45 C and
added to three 250 mL plastic boxes, then cooled further until solidification (each box contained 200 mL
solution). An 88.75 µL volume of the Mcl working solution was mixed with 1.2 L 0.3% agar medium
solution to obtain an agar medium with 0.25
medium were added to three 250 mL plastic boxes at 45 C and cooled until solidification. Using the
µ
M
Mcl. Three volumes of 200 mL 0.25
µ
M
Mcl agar
◦
same method, solidified agar media (without metolachlor) containing 8 mg/L of the target compounds
(
2a
–
k
,
3a
Agar media containing 0.25
was also prepared. 2a 3a 4a–k
–k, and 4a–k) were prepared to test the phytotoxicity of target compounds on rice seedlings.
µM
Mcl
, and
,
2a
D
–
k
,
3a
–k, 4a–k, and the positive control dichlormid (D)
–
k,
–
k
,
(each 2.4 g) were dissolved in separate 1 mL volumes
of acetone, and then each was diluted to 12.5 mL using deionized water containing 0.5% Tween 80
to obtain 192 g/ L standard solutions. A 40 L volume of each standard solution was added to
separate volumes of 1.2 L 0.25 Mcl agar medium to obtain an agar media containing 8 mg/L of
µ
µ
µ
µM
each compound. Three 200 mL volumes of each agar medium were poured and set in three 250 mL
plastic boxes, respectively.

Molecules 2020, 25, 4986
14 of 18
Before shoot emergence, uniformly germinated rice seedlings were planted in different 0.3%
agar mediums: 0.25 Mcl; 8 mg/L of compounds 2a 3a , and 4a ; 8 mg/L compounds 2a
4a , and with 0.25
µM
–
k,
–
k
–
k
–k,
3
a
–
k
,
–
k
D
µ
M
Mcl and no additional compounds (non-treatment, control check).
◦
Fifty seeds were added to each plastic box and incubated for 14 h at 30 C under a grow light (intensity
2
◦
1
10–130
to herbicide safener activity (plant height, root length, and fresh weight) were measured after 7-day
treatment. The phytotoxicity indexes of compounds 2a 3a , and 4a on rice seedlings and their
µ
E/m /s), followed by a 10 h dark photoperiod at 25 C. The various growth indexes related
–k
,
–k
–k
herbicide safening indexes were further used for the analysis of structure-activity relationship (SAR)
according to the following equations:
Plant height relative value = (plant height under each treatment)/(the average
(1)
height of non-treatment) × 100%
Root length relative value = (root length under each treatment)/(the average root
length of non-treatment) × 100%
Fresh weight relative value = (fresh weight under each treatment)/(the average
fresh weight of non-treatment) × 100%
(
(
2)
3)
Compounds deemed to have high herbicide safener activities based on plant height were screened
further for herbicide safener activities in subsequent experiments at lower concentrations (0.25 mg/L,
0
.5 mg/L, 1 mg/L, 2 mg/L, and 4 mg/L). All experiments on herbicide safener activity were carried out
in three replications.
3
.4. Enzyme Activity Assays
Rice seedling samples were collected after the 7-day treatments with 0.25
the combination of 8 mg L compounds 2k, 4k, and dichlormid with 0.25 µM metolachlor, and control
µM metolachlor,
−1
check (CK, no-treatment). 0.1 g of rice seedling tissues from the above treatments were first homogenized
via a high-speed electric homogenizer, then filtered, and a 1 mL enzyme extraction solution was
added. After centrifugation (12,000 r/min for 10 min), the supernatant was collected for use in enzyme
activity assay.
Glutathione S-transferase (GST: EC 2.5.1.18) activity was examined by calculating the quantity
of glutathione conjugate (composed of glutathione (GSH) and chlorodinitrobenzene (CDNB)) at
3
40 nm [56]. The activity was expressed as the enzyme unit nmol/min/g FW. Superoxide dismutase (SOD:
EC 1.15.1.1) activity was measured through the method reported previously by calculating reduction
inhibition of WST-1 (5-(2,4-disulfophenyl)-2-(4-iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium inner
salt sodium salt (1:1)) at 450 nm [57], and the activity was articulated as the unit U/g FW. Catalase (CAT:
EC 1.11.1.6) activity was evaluated at 510 nm by estimating the breakdown of H O , and the CAT
2
2
activity was expressed as the unit nmol/min/g FW [58]. Peroxidase (POD: EC 1.15.1.1) activity was
measured at 470 nm by calculating the oxidation of O-dianisidine in the presence of H O , and the
2
2
enzyme activity was articulated as
activity was measured at 420 nm and expressed as the unit
of nicotinamide adenine dinucleotide phosphate (NADPH) [60].
∆
OD470/min/g FW [59]. Polyphenol oxidase (PPO: EC 1.10.3.1)
∆
OD420/min/g FW, based on the oxidation
3
.5. Acute Toxicities of 2k, 4k, and Dichlormid to Zebrafish Embryos
The acute toxicities (96h) of 2k 4k, and dichlormid to zebrafish embryos were evaluated via
the method reported by Chen et al. [61]. Zebrafish embryos (2 h post-fertilization) were randomly
transferred into 2 mL of test solutions with varying concentrations of 2k 4k, and dichlormid in 24-well
plates. Experiments were carried out to test the acute toxicity (median lethal concentration, LC50 value)
of 2k 4k, and dichlormid at 96 h. The concentrations of 2k and 4k were 5, 10, 20, 40, and 80 mg/L,
while that of dichlormid were 2.5, 5, 10, 20, 40, and 80 mg/L, respectively. For each concentration,
,
,
,

Molecules 2020, 25, 4986
15 of 18
6
0 embryos were used, and all experiments were done in three replications. Experimental plates were
covered with transparent lids to prevent evaporation and transferred to an incubator maintained
◦
at 27
±
1 C with a 14:10 h light/dark photoperiod. The number of dead individuals and the state
+
of embryonic development were examined daily. Reconstituted water containing 0.75 mmol/L Na ,
.5 mmol/L Mg , 0.074 mmol/L K , and 2 mmol/L Ca was used to prepare all test solutions
2
+
+
2+
0
and a control solvent of 0.01% acetone (control check, v/v). The water solubility of 2k and 4k was
evaluated by the reported method [62
,63], and the solubility of 2k and 4k was 1.23 g/L and 3.13 g/L at
◦
2
5 C, respectively.
3
.6. Statistical Analysis
All data were analyzed by one-way analysis of variance (ANOVA; general linear models on
Origin 8.0 procedure). The Bonferroni’s test was used for mean separation if the ANOVA test indicated
significance. A value of p < 0.05 was considered statistically significant.
4
. Conclusions
In summary, 33 N-alkyl amide derivatives (2a
and saturated and unsaturated fatty acids through acylation and condensation via the scaffold hopping
–k, 3a–k, and 4a–k) were synthesized from amines
1
13
approach. The chemical structures of all target compounds were well confirmed by H-NMR, C-NMR,
and HRMS. A primary test by the agar method demonstrated that most of the target compounds
could protect rice seedlings from injury caused by the pesticide metolachlor. Notably, compounds 2k
and 4k displayed excellent herbicide safener activities on plant height and showed relatively similar
activities to the commercialized compound dichlormid. Further, we showed that compounds 2k and
4k had higher GST, SOD, CAT, POD, and PPO enzyme activities in treated rice seedlings relative to
the metolachlor treatment. Results from the current work revealed that compounds 2k and 4k had
good safener activities toward rice and are, thus, promising herbicide safener candidates for further
structural optimization to identify novel herbicide safeners for chloroacetanilide herbicides that are
eco-friendly to the environment.
Supplementary Materials: H NMR and 13C NMR spectrums of target compounds 2a–k, 3a–k, and 4a–k.
1
Author Contributions: Conceptualization, X.D. and L.B.; methodology, X.D.; software, W.Z.; validation, W.Z.;
formal analysis, W.Z.; investigation, W.Z.; resources, Q.Z.; data curation, W.Z.; writing—original draft preparation,
X.D.; writing—review and editing, X.D.; visualization, X.D. and Y.D.; supervision, Y.Z.; project administration,
L.B.; funding acquisition, L.B. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (No. 31772182).
Conflicts of Interest: The authors declare no conflict of interest.
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