Synthesis and biological evaluation of norcantharidin derivatives possessing an aromatic amine moiety as antifungal agents

Article abstract of DOI:10.3390/molecules201219782

Based on the structure of naturally produced cantharidin, different arylamine groups were linked to the norcantharidin scaffold to provide thirty six compounds. Their structures were confirmed by melting point, ¹H-NMR, ¹³C-NMR and HRMS-ESI studies. These synthetic compounds were tested as fungistatic agents against eight phytopathogenic fungi using the mycelium growth rate method. Of these thirty six derivatives, seven displayed stronger antifungal activity than did norcantharidin, seven showed higher activity than did cantharidin and three exhibited more significant activity than that of thiabendazole. In particular, 3-(3′-chloro-phenyl)carbamoyl norcantharidate II-8 showed the most significant fungicidal activity against Sclerotinia fructigena and S. sclerotiorum, with IC₅₀ values of 0.88 and 0.97 μg/mL, respectively. The preliminary structure-activity relationship data of these compounds revealed that: (1) the benzene ring is critical for the improvement of the spectrum of antifungal activity (3-phenylcarbamoyl norcantharidate II-1 vs norcantharidin and cantharidin); (2) among the three sites, including the C-2′, C-3′ and C-4′ positions of the phenyl ring, the presence of a halogen atom at the C-3′ position of the benzene ring caused the most significant increase in antifungal activity; (3) compounds with strongly electron-drawing or electron-donating groups substitutions were found to have a poor antifungal activity; and (4) compared with fluorine, bromine and iodine, chlorine substituted at the C-3′ position of the benzene ring most greatly promoted fungistatic activity. Thus, compound II-8 has emerged as new lead structure for the development of new fungicides.

Full text of DOI:10.3390/molecules201219782

Article
Synthesis and Biological Evaluation of
Norcantharidin Derivatives Possessing an Aromatic
Amine Moiety as Antifungal Agents
Yang Wang 1,†, Wenbo Sun 2,†, Shunqing Zha 1, Huan Wang 2 and Yalin Zhang 2,
*
Received: 11 November 2015 ; Accepted: 30 November 2015 ; Published: 2 December 2015
Academic Editor: Derek J. McPhee
1
State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A & F
University, Yangling 712100, China; wangyang2006@nwsuaf.edu.cn (Y.W.); zsq2623@163.com (S.Z.)
Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education,
Northwest A & F University, Yangling 712100, China; wbsun06@163.com (W.S.);
huanwang1107@gmail.com (H.W.)
2
*
†
Correspondence: yalinzh@nwsuaf.edu.cn; Tel./Fax: +86-29-8709-2190
These authors contributed equally to this work.
Abstract: Based on the structure of naturally produced cantharidin, different arylamine
groups were linked to the norcantharidin scaffold to provide thirty six compounds. Their
structures were confirmed by melting point, 1H-NMR, 13C-NMR and HRMS-ESI studies. These
synthetic compounds were tested as fungistatic agents against eight phytopathogenic fungi
using the mycelium growth rate method. Of these thirty six derivatives, seven displayed
stronger antifungal activity than did norcantharidin, seven showed higher activity than did
cantharidin and three exhibited more significant activity than that of thiabendazole. In particular,
1
3-(3 -chloro-phenyl)carbamoyl norcantharidate II-8 showed the most significant fungicidal
activity against Sclerotinia fructigena and S. sclerotiorum, with IC50 values of 0.88 and 0.97 µg/mL,
respectively. The preliminary structure-activity relationship data of these compounds revealed
that: (1) the benzene ring is critical for the improvement of the spectrum of antifungal activity
(3-phenylcarbamoyl norcantharidate II-1 vs norcantharidin and cantharidin); (2) among the three
1
1
1
sites, including the C-2 , C-3 and C-4 positions of the phenyl ring, the presence of a halogen atom
1
at the C-3 position of the benzene ring caused the most significant increase in antifungal activity;
(3) compounds with strongly electron-drawing or electron-donating groups substitutions were
found to have a poor antifungal activity; and (4) compared with fluorine, bromine and iodine,
1
chlorine substituted at the C-3 position of the benzene ring most greatly promoted fungistatic
activity. Thus, compound II-8 has emerged as new lead structure for the development of new fungicides
.
Keywords: cantharidin; natural product; structural modification; arylamine; fungicidal activity
1. Introduction
Fungal pathogens are the primary causes of both plant diseases and postharvest losses [1,2], and
thus contribute to severe damage to global crop production [3]. To guard against fungal pathogens,
one traditional approach is to employ synthetic fungicides, which are both economical and efficient,
and have played an indispensable role in nourishing more people throughout human history.
Unfortunately, drug resistance, environmental hazards and many other drawbacks have emerged
along with this fungicide utilization [4,5]. This requires that novel antifungal agents continue to be
discovered. It is well-known that insect secondary metabolites result from their adaptation to the
environments during the long period of evolution in insects, such as for defense against predation
Molecules 2015, 20, 21464–21480; doi:10.3390/molecules201219782
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Molecules 2015, 20, 21464–21480
or infection, and pesticides produced from insect secondary metabolites may result in less or slower
resistance development and lower pollution [6]. Hence, insecticides of natural source have been
considered as attractive alternatives to synthetic agrochemicals for pest management [7]. Bio-based
insecticides, such as nereistoxin, nicotine, pyrethrum, and neem extracts, are made by organisms as
defense against insects [8].
Cantharidin (CTD, Figure 1A), a naturally occurring terpene, is the main secondary metabolite
isolated from the bodies of the blister beetles, including Mylabris cichorii, M. phalerata and Epicauta
chinensis [9,10]. Besides its use as the lead compound for the preparation of potent anticancer drugs,
such as its analogues sodium cantharidinate and norcantharidin [11–14], CTD also shows interesting
antifungal and insecticidal activities [15–18].
Figure 1. The structures of cantharidin (A) and norcantharidin (B).
Norcantharidin (NCTD, Figure 1B), a demethylated analogue of CTD, is well known as a strong
inhibitor of protein serine/threonine phosphatases (PSPs) [19], a broad class of PSPs associated with
signaling and control of numerous cellular processes in many organisms [20]. The biological activities
associated with NCTD are derived from its abilities to inhibit the family of PSPs. Furthermore, the
catalytic domain of all PSP subfamilies is highly conserved in insects, plants, phytopathogenic fungi
and all the other eukaryotes [21]. NCTD, as the strong inhibitor, binds to a hydrophobic pocket of the
PSP active site [22]. The structural similarity between CTD and NCTD has been apparent to animal
scientists and similar mechanisms of action on animals have been confirmed [23]. Previous studies
by our group demonstrated that both in vivo and in vitro, there are significant inhibitory effects from
CTD, NCTD and their analogues on PSPs of Plutella xylostella, suggesting that their modes of action
may be related to impeding insect PSPs activity [24]. More recently, strong evidence indicates that the
commercial herbicide mode of action of endothall, an analogue of NCTD, was intimately related to
the inhibition of PSPs [25].
Meanwhile, as illustrated in Figure 2, our previous investigation on the structure-activity
relationship (SAR) of NCTD and its anhydride modified derivatives against P. xylostella indicated
that the structures of oxygen bridge and carboxyl were essential for the biological activity, and that
the improvement of bioactivity required a reasonable R group, the combination of both aliphatic
amide and aromatic amide moieties. And the type of substituent Y, substituted on the phenyl ring,
was also critical for the improvement of insecticidal activity [26].
Figure 2. Design of the title compounds.
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Molecules 2015, 20, 21464–21480
To date, few attempts have been made to develop a biorational fungicide from NCTD via
chemical modification. Encouraged by the abovementioned results and in continuation of our
program aimed at the discovery and development of natural-product-based fungicidal agents, in
this study, thirty six aromatic amine derivatives of NCTD modified in the anhydride ring were
synthesized (Scheme 1), and their biological activities against eight phytopathogenic fungi were
evaluated using the mycelium growth rate method [27]. Additionally, their preliminary SAR studies
were also described.
R
O
9HN
6
5
8
1
2
THF
O
+
H2N R
O
O
O
TEA, rt
7
4
OH
3
O
O
II (1~36)
I
F
F
3' 2'
1'
F
4'
5' 6'
1
2
8
3
4
5
6
Cl
Cl
Br
Br
Cl
Br
7
9
10
11
12
I
I
OCH3
I
HO
NC
HOOC
13
19
14
20
15
21
16
22
17
23
18
24
H3CO
NO2
O2N
H3CO
F3CO
Cl
Br
H3CO
H3CO
OCH3
O2N
Cl
Br
I
H3CO
25
31
26
27
28
29
30
O2N
H
N
S
N
S
N
N
N
32
33
34
35
36
Scheme 1. The Synthesis and Structures of Compounds II (1–36).
2. Results and Discussion
2.1. Chemistry
As shown in Scheme 1, the target compounds II were synthesized by the aminolysis reaction of
NCTD I and various halogen aromatic amines in the presence of triethylamine as the binding acid
agent, and synthesized via decomposing the anhydride ring of NCTD I by a halogenated aniline with
different electronegativity [28].
2.2. Antifungal Activity
Preliminary in vitro screening results of the title compounds for antifungal activities against eight
fungi at the concentration of 50 µg/mL are listed in Table 1.
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Molecules 2015, 20, 21464–21480
Table 1. Preliminary antifungal activities of compounds at 50 µg/mL.
Values of Inhibition Rate (%) to Eight Pathogens
Compd.
Botryosphaeria
berengeriana
Sclerotinia
fructigena
Glomerella
cingulate
Alternaria
alternate
Sclerotinia
sclerotiorum
Cochliobolus
sativum
Valsa mali
Alternaria solani
II-1
II-2
II-3
II-4
II-5
II-6
II-7
II-8
II-9
68.42 ˘ 1.99d *
0.66 ˘ 0.06q
18.42 ˘ 0.06mn
74.99 ˘ 1.30c
83.56 ˘ 1.05b
41.45 ˘ 0.47e
34.21 ˘ 1.00f
86.85 ˘ 1.00a
32.88 ˘ 3.85f
25.01 ˘ 3.06g
80.92 ˘ 1.26b
20.39 ˘ 1.04h
15.14 ˘ 1.23i
65.79 ˘ 1.00d
3.29 ˘ 1.16j
68.64 ˘ 0.65e
1.27 ˘ 0.03qr
11.02 ˘ 0.10l
69.90 ˘ 1.17e
87.70 ˘ 0.91c
39.39 ˘ 1.61f
22.86 ˘ 1.85h
91.54 ˘ 1.33b
22.01 ˘ 2.89h
18.18 ˘ 3.79i
81.77 ˘ 0.90d
15.24 ˘ 3.23i
10.99 ˘ 2.47j
66.51 ˘ 1.95e
´5.12 ˘ 3.86k
0.85 ˘ 0.04pqr
0.42 ˘ 0.06qr
6.36 ˘ 0.06mn
10.17 ˘ 0.06l
2.12 ˘ 0.10pqr
10.59 ˘ 0.06l
0.00 ˘ 0.066r
0.00 ˘ 0.00r
72.20 ˘ 0.66e
0.53 ˘ 0.06qr
18.72 ˘ 0.06m
76.48 ˘ 1.63d
85.03 ˘ 1.79bc
49.21 ˘ 1.37f
45.47 ˘ 2.26g
100.00 ˘ 0.00a
28.36 ˘ 2.69h
23.00 ˘ 2.51i
83.42 ˘ 2.49c
17.13 ˘ 2.59j
22.46 ˘ 1.62i
70.60 ˘ 2.19e
1.07 ˘ 0.92k
8.56 ˘ 0.06p
´0.53 ˘ 0.06r
0.00 ˘ 0.00qr
2.67 ˘ 0.06qr
0.53 ˘ 0.06qr
11.76 ˘ 0.10o
8.02 ˘ 0.11p
9.09 ˘ 0.11p
18.18 ˘ 0.10m
12.30 ˘ 0.06o
48.13 ˘ 0.06g
39.57 ˘ 0.06i
39.04 ˘ 0.06i
36.36 ˘ 0.06j
14.97 ˘ 0.10n
17.11 ˘ 0.06m
6.42 ˘ 0.06p
28.88 ˘ 0.06k
62.35 ˘ 0.99f
5.38 ˘ 0.05tu
6.99 ˘ 0.06st
80.66 ˘ 1.18d
90.34 ˘ 2.64b
55.40 ˘ 1.36g
24.74 ˘ 0.83h
92.96 ˘ 2.69a
24.18 ˘ 1.08h
21.48 ˘ 1.28i
84.97 ˘ 1.47c
18.25 ˘ 1.37j
12.35 ˘ 0.59k
60.77 ˘ 0.78f
5.37 ˘ 0.86l
65.52 ˘ 1.06de
9.66 ˘ 0.06no
11.72 ˘ 0.06n
70.34 ˘ 1.41c
84.82 ˘ 1.30a
41.38 ˘ 2.14g
38.62 ˘ 1.04g
86.90 ˘ 2.32a
30.34 ˘ 3.13h
23.44 ˘ 2.27i
78.63 ˘ 2.28b
22.07 ˘ 3.14i
17.22 ˘ 4.17j
62.76 ˘ 2.13e
3.44 ˘ 1.18k
76.39 ˘ 0.92e
23.61 ˘ 0.06p
20.17 ˘ 0.00q
86.69 ˘ 0.85d
92.27 ˘ 0.06c
46.34 ˘ 2.29f
35.19 ˘ 1.78g
100.00 ˘ 0.00a
26.60 ˘ 2.79i
30.46 ˘ 3.04h
96.13 ˘ 1.32b
20.59 ˘ 2.45j
15.87 ˘ 1.37k
73.80 ˘ 3.43e
8.15 ˘ 1.43l
61.10 ˘ 0.96e
1.52 ˘ 0.10u
1.01 ˘ 0.06u
76.77 ˘ 0.26c
83.35 ˘ 1.16b
47.46 ˘ 1.21f
30.29 ˘ 0.92g
89.90 ˘ 0.79a
28.27 ˘ 1.83hi
18.17 ˘ 2.45j
79.83 ˘ 1.81bc
25.23 ˘ 1.87i
16.16 ˘ 2.31j
58.60 ˘ 1.33e
11.10 ˘ 6.18k
2.02 ˘ 0.10u
7.58 ˘ 0.12s
67.01 ˘ 0.59c
7.98 ˘ 0.06qr
10.11 ˘ 0.06pqr
66.50 ˘ 0.30c
81.39 ˘ 0.38b
51.59 ˘ 0.76e
35.06 ˘ 1.80f
91.50 ˘ 0.66a
17.53 ˘ 0.90j
II-10
II-11
II-12
II-13
II-14
II-15
II-16
II-17
II-18
II-19
II-20
II-21
II-22
II-23
II-24
II-25
II-26
II-27
II-28
II-29
II-30
II-31
II-32
II-33
21.21 ˘ 2.53i
83.56 ˘ 1.80b
13.78 ˘ 1.91k
9.54 ˘ 1.22l
63.86 ˘ 1.01d
´2.71 ˘ 1.98m
5.85 ˘ 0.10s
9.21 ˘ 0.10p
0.66 ˘ 0.06q
1.97 ˘ 0.06q
0.66 ˘ 0.06q
0.66 ˘ 0.06q
1.32 ˘ 0.10q
24.34 ˘ 0.06k
19.74 ˘ 0.06m
25.00 ˘ 0.10q
23.03 ˘ 0.10kl
32.89 ˘ 0.10j
36.18 ˘ 0.06hi
8.55 ˘ 0.06p
30.26 ˘ 0.06j
0.66 ˘ 0.06q
15.13 ˘ 0.10o
3.29 ˘ 0.10 q
0.66 ˘ 0.06q
9.14 ˘ 0.03rs
0.54 ˘ 0.06wx
0.00 ˘ 0.00x
10.34 ˘ 0.06no
1.38 ˘ 0.06pq
8.28 ˘ 0.06no
15.86 ˘ 0.06m
2.07 ˘ 0.06pq
20.69 ˘ 0.06ijk
17.93 ˘ 0.06jklm
0.69 ˘ 0.10pq
28.28 ˘ 0.06h
21.38 ˘ 0.10ij
35.86 ˘ 0.10g
20.69 ˘ 0.06ijk
38.62 ˘ 0.10fg
40.00 ˘ 0.06f
10.34 ˘ 0.16no
´1.38 ˘ 0.06q
6.90 ˘ 0.06o
6.87 ˘ 0.06t
0.43 ˘ 0.08u
0.00 ˘ 0.06u
0.00 ˘ 0.06u
0.00 ˘ 0.06u
13.30 ˘ 0.06s
18.45 ˘ 0.06q
13.73 ˘ 0.10t
19.31 ˘ 0.06q
7.73 ˘ 0.06u
40.34 ˘ 0.06k
42.96 ˘ 0.06j
42.06 ˘ 0.00jk
36.48 ˘ 0.06l
26.18 ˘ 0.06no
12.02 ˘ 0.06s
7.30 ˘ 0.00t
12.23 ˘ 0.10nopq
0.00 ˘ 0.00t
0.00 ˘ 0.00u
2.53 ˘ 0.00u
0.00 ˘ 0.00u
21.72 ˘ 0.06lm
22.73 ˘ 0.06kl
2.53 ˘ 0.03u
7.07 ˘ 0.03st
3.54 ˘ 0.03tu
33.33 ˘ 0.12op
29.80 ˘ 0.06ij
16.67 ˘ 0.10nop
20.20 ˘ 0.06lmn
12.12 ˘ 0.10qr
7.07 ˘ 0.10st
18.69 ˘ 0.06mno
6.57 ˘ 0.06st
11.83 ˘ 0.05pq
0.00 ˘ 0.00x
11.70 ˘ 0.06opq
0.00 ˘ 0.00t
15.59 ˘ 0.06o
12.90 ˘ 0.05p
3.23 ˘ 0.03uv
20.43 ˘ 0.06m
17.20 ˘ 0.05no
28.49 ˘ 0.06h
22.04 ˘ 0.06jkl
23.66 ˘ 0.06ijk
21.51 ˘ 0.06kl
4.30 ˘ 0.06tuv
4.84 ˘ 0.00tuv
5.91 ˘ 0.06tu
2.69 ˘ 0.06vw
22.87 ˘ 0.06hi
12.23 ˘ 0.03nopq
11.17 ˘ 0.06opq
15.43 ˘ 0.10lm
6.38 ˘ 0.11rs
5.08 ˘ 0.06n
2.54 ˘ 0.11pq
18.22 ˘ 0.06k
15.25 ˘ 0.06j
6.78 ˘ 0.06o
0.42 ˘ 0.06qr
0.00 ˘ 0.06r
37.23 ˘ 0.06f
14.89 ˘ 0.06lmn
12.77 ˘ 0.06mnop
12.77 ˘ 0.10mnop
18.62 ˘ 0.10jk
12.23 ˘ 0.11nopq
22.34 ˘ 0.06hi
´1.60 ˘ 0.07t
0.00 ˘ 0.06r
8.05 ˘ 0.06m
0.00 ˘ 0.11r
´0.69 ˘ 0.05q
23.61 ˘ 0.08p
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Molecules 2015, 20, 21464–21480
Table 1. Cont.
Values of Inhibition Rate (%) to Eight Pathogens
Compd.
Botryosphaeria
berengeriana
Sclerotinia
fructigena
Glomerella
cingulate
Alternaria
alternate
Sclerotinia
sclerotiorum
Alternaria
solani
Cochliobolus
sativum
Valsa mali
II-34
II-35
II-36
36.84 ˘ 0.10h
30.26 ˘ 0.15g
61.93 ˘ 0.10e
0.42 ˘ 0.06qr
2.97 ˘ 0.06o
72.88 ˘ 0.06e
34.31 ˘ 1.69g
38.54 ˘ 2.68f
100.00 ˘ 0.00a
30.48 ˘ 0.06k
21.39 ˘ 0.10l
68.98 ˘ 0.15f
86.64 ˘ 0.80b
100.00 ˘ 0.00a
100.0 ˘ 0.00a
0.54 ˘ 0.06x
5.91 ˘ 0.06tu
62.37 ˘ 0.06e
17.72 ˘ 1.21j
10.19 ˘ 1.61k
66.67 ˘ 0.00e
0.69 ˘ 0.06pq
0.00 ˘ 0.03pq
61.38 ˘ 0.03d
20.00 ˘ 1.10ij
67.59 ˘ 1.06cd
47.58 ˘ 1.60f
69.53 ˘ 0.06h
0.00 ˘ 0.00u
77.25 ˘ 0.06f
24.46 ˘ 1.13i
84.54 ˘ 1.39d
100.00 ˘ 0.00a
13.13 ˘ 0.06pqr
0.51 ˘ 0.06u
62.63 ˘ 0.06e
1.98 ˘ 2.24l
15.43 ˘ 0.10lm
10.11 ˘ 0.03pqr
65.43 ˘ 0.03cd
29.75 ˘ 1.27g
68.05 ˘ 1.29c
24.99 ˘ 1.17h
Norcantharidin 88.17 ˘ 1.87a
Cantharidin
88.16 ˘ 1.39a
70.20 ˘ 0.84d
15.14 ˘ 1.18j
Thiabendazole 86.84 ˘ 1.22a
* The differences between data with different letters within a column are significant for the same tested fungus (p < 0.05) with respect to TBZ, cantharidin, norcantharidin and these
synthetic compounds with a halogenated benzene moiety.
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Molecules 2015, 20, 21464–21480
The results in Table 1 indicate that seven of the synthetic derivatives displayed significant
activities (61.1%–100% inhibitory rate) against all eight tested fungi at a concentration of 50 µg/mL.
On the contrary, another twenty nine derivatives showed lower activity at the same concentration.
Here, the inhibition rates were for only given as IC50 values for further comparison.
Encouraged by these preliminary findings, we planned further SAR studies on the title
compounds. We determined their IC50 values by the mycelial growth inhibitory rate method. As
shown in Table 2, the tested compounds presented different fungicidal activity against the eight plant
pathogenic fungi, superior to the corresponding parent compound NCTD in some cases, and they
were the same as or more active than thiabendazole (TBZ) against some of the tested fungi. Of
1
these compounds, 3-(3 -chlorophenyl)carbamoyl norcantharidate II-8 exhibited the most significant
activity on all the eight fungi. As illustrated in Figure 3, compound II-8 showed much better activity
than that of TBZ, NCTD and CTD. Notably, compound II-8 showed excellent antifungal properties
against Sclerotinia fructigena and S. sclerotiorum, with IC50 values of 0.88 and 0.97 µg/mL, respectively.
Figure 3. IC50 of compound II-8, CTD, NCTD and TBZ against the eight tested fungi.
2.3. SAR
2.3.1. Effect of Introducing the Benzene Ring on Fungistatic Activity
Illustrated in Table 2, NCTD, without a benzene ring, exhibited low fungistatic activity at
the concentration of 50 µg/mL against the tested strains. Introducing one benzene (2a) caused
a significant increase in fungistatic activity against the plant-pathogenic fungi Botryosphaeria
berengeriana, S. fructigena, Glomerella cingulate, Alternaria alternate, S. sclerotiorum, A. solani and
Cochliobolus sativum, with IC50 values of 31.2568, 14.6778, 36.3526, 42.2537, 7.2482, 30.1335 and
22.7788 µg/mL, respectively. At the same time, as seen in Table 2, compared with the fungicidal
activities of CTD against B. berengeriana and G. cingulate, the compound II-1 also showed almost 5-fold
more fungistatic activity than CTD, respectively. On the contrary, the introduction of a heterocyclic
ring gave us a negative contribution to fungistatic activity.
2.3.2. Effect of Position Substituted on the Benzene Ring on Fungistatic Activity
1
Substituting a halogen at the C-3 position of the benzene ring (i.e., compounds II-5, II-8 and
II-11) improved the fungicidal activity in some cases. It was also clear that the site substituted on
1
the phenyl ring plays an important role in activity, as compounds substituted at the C-4 position
1
(II-6, II-9 and II-12) and C-2 position (II-4, II-7 and II-10) were all found to be less active than
their analogues.
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Molecules 2015, 20, 21464–21480
Table 2. Antifungal activity of some compounds against eight pathogens *.
IC50 (µg/mL) (CI 95%) **
Compd.
V. m.
B. b.
S. f.
G. c.
A. a.
S. s.
A. s.
C. s.
32.0800
(26.1510–39.3533)
27.8016
(22.1312–34.9249)
20.1058
(14.8666–27.1914)
11.3756
(7.2790–17.7778)
17.0896
(11.9965–23.3450)
47.7810
(39.0992–58.39.4)
17.1862
(9.2272–32.0104)
21.9320
(13.3453–36.0436)
0.5191
31.2568
(23.7127–41.2012)
24.8446
(18.2585–33.8063)
19.8375
(14.7821–26.6217)
13.6528
(9.8721–18.8814)
17.0640
(12.6223–23.0689)
37.3895
(28.8394–48.4746)
141.8133
(61.1287–328.9946)
147.4225
(46.8714–463.6810)
15.5378
14.6778
(10.7872–19.9716)
10.6665
(7.6148–14.9412)
2.0791
(1.3098–3.3003)
0.8805
(0.4243–1.8275)
2.3717
(1.4367–3.9152)
21.9666
(16.3757–29.4664)
17.1673
(10.0952–29.1937)
2.2175
(0.6116–12.0751)
3.1998
36.3526
(29.1302–45.3658)
16.7764
(11.7587–23.9352)
11.2505
(6.9283–18.2691)
7.7364
(3.8335–15.6128)
9.1071
(5.1797–16.0125)
57.0963
(45.2452–72.0517)
465.1719
42.2537
(33.7577–52.8879)
31.4258
(24.6239–40.1066)
18.1002
(13.1268–24.9579)
13.8916
(9.3377–20.6664)
22.1465
(16.7792–29.2308)
49.1546
(39.1730–61.6795)
394.9566
7.2482
(2.9912–17.5637)
4.2858
(1.6648–11.0334)
3.4556
(1.6958–7.0418)
0.9698
(0.4790–1.9633)
2.1783
(0.9142–5.1904)
9.6192
(4.2886–21.5754)
54.5899
(32.0147–93.0839)
14.0622
(8.0013–24.7142)
1.2947
30.1335
(23.3387–38.9065)
14.0938
(9.1043–21.8178)
10.4753
(6.7490–16.2590)
5.1863
(2.5133–10.7020)
12.1081
(7.9591–18.4200)
34.8290
(27.5093–44.0964)
594.2606
(211.1064–1672.8327)
28.5223
(18.6015–43.7340)
1268.5889
22.7788
(16.0894–32.2493)
10.4051
(12.9130–24.7109)
10.4051
(6.4109–16.8880)
7.5908
(4.4564–12.9298)
12.9298
II-1
II-4
II-5
II-8
II-11
(8.9552–18.6685)
29.8409
(16.0894–32.2493)
II-14
Norcantharidin
Cantharidin
TBZ
NA ***
(86.5242–2500.8598) (73.9654–2108.9689)
155.0286
(76.7546–313.1261)
22.3806
14.0428
(5.6729–34.7622)
47.8696
18.3900
(8.3854–40.3309)
621.7874
(0.0218–12.3413)
(8.8501–27.2793)
(1.2133–8.4391)
(10.4428–47.9653)
21.2217–107.9790
(0.6661–2.5165)
(110.6555–1453.4930) 60.1464–6427.9804
* V. m.: Valsa mali, B. b.: Botryosphaeria berengeriana, S. f.: Sclerotinia fructigena, G. c.: Glomerella cingulate, A. a.: Alternaria alternate, S. s.: Sclerotinia sclerotiorum, A. s.: Alternaria solani,
C. s.: Cochliobolus sativum. ** CI 95%: Confidence interval at 95% probability (µg/mL). *** NA: Not Available.
21470

Molecules 2015, 20, 21464–21480
2.3.3. Effect of Various Substituents on the Benzene Ring on Fungistatic Activity
The presence of different halogen atoms on the benzene ring resulted in various effects on
1
fungistatic activity against the eight fungi tested. As can be seen from Table 2, at the C-3 position
of the benzene ring, introduction of the chlorine atom (II-8) produced a more significant increase
in the fungistatic activity against all eight fungi tested than introductions of a fluorine atom (II-5)
or a bromine atom (II-11). In contrast, the introduction of an iodine atom on the benzene ring
(II-14) resulted in a negative influence on fungistatic activity compared with compound II-1 with
no substitution on the phenyl ring.
Compounds with substitutions of strongly electron-drawing (CN, compound II-22; NO2,
compounds II-23, 24, 25) or electron-donating (CH3, compound II-2; CH2CH3, compound II-3;
OCH3, compounds II-18, 19, 20) groups were found to have a poor antifungal activity. In order
to draw firm conclusions about the significance of these data, compounds containing one phenyl ring
substituted by two methoxy groups in different positions (compounds II-29, 30) were also tested, and
showed the same poor activity. The nitro group was tested in other positions in combination with
a methyl group (compound II-31), but only poor spectrum and antifungal activity were detected.
Further aromatic groups were also evaluated. Compounds with the phenyl moiety replaced with
several kinds of heterocycle produced relatively poor levels of antifungal activity compared to the
corresponding compound II-1.
3. Experiment Section
3.1. General Information
Cantharidin (CTD, ě98%) was isolated from M. phalerata (Chinese blister beetle), bought from
the Chinese herbal medicine market in Xian, China. Norcantharidin (NCTD, ě98%) was purchased
from Alfa Aesar Chemical Co. Ltd., (Haverhill, MA, USA). Dimethyl sulfoxide (DMSO, ě99%) was
obtained from J & K China Chemical Ltd. (Beijing, China). The fungicide thiabendazole (TBZ,
ě99.1%) was purchased from Sigma-Aldrich Trading Co. Ltd. (Shanghai, China). All reagents and
solvents were of reagent grade or purified according to standard methods before use. Analytical
thin-layer chromatography (TLC) was performed with silica gel plates using silica gel 60 GF254
(Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Silica gel column chromatography was
performed with 200–300 mesh silica gel (Qingdao Haiyang Chemical Co., Ltd.).
Melting points were determined on a WRS-2 apparatus equipped with a microcomputer
(Shanghai Precision & Scientific Instrument Co. Ltd., Shanghai, China) and are uncorrected. Proton
nuclear magnetic resonance (1H-NMR) spectra and carbon nuclear magnetic resonance (13C-NMR)
spectra were recorded at 500 and 125 MHz, respectively, on a Bruker Avance III 500 MHz NMR
spectrometer (Karlsruhe, Germany) in DMSO-d6 using tetramethylsilane (TMS) as the internal
standard. High-resolution mass spectrometry (HRMS) was carried out using a Bruker micrOTOF
focus II instrument.
3.2. Synthesis of the Title Compounds II(1–36)
To a solution of NCTD (1, 1.0 g, 5.95 mmol) dissolved in tetrahydrofuran (THF, 10 mL)
accompanied by triethylamine (TEA, 0.5 mL) as the acid-binding agent was added the corresponding
aromatic amine (1 equiv., 5.95 mmol). When the reaction was complete after 14 h as checked
by TLC analysis, the solution was concentrated under reduced pressure and diluted with acetone
(100 mL). The resulting filter cake was either recrystallized from methanol or purified by column
chromatography (MeOH/CH2Cl2, 1: 4, v/v) to afford the desired products II (1–36). The yields,
physical properties, 1H-NMR, 13C-NMR, and HRMS-ESI of the target compounds II were as follows:
3-(Phenylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-1). Yield 76%, white solid, mp:
˝
1
164–165 C; H-NMR δ: 1.47–1.68 (m, 4H, H-1, 2), 2.93–3.12 (m, 2H, H-4, 5), 4.61–4.81 (m, 2H, H-3,
21471

Molecules 2015, 20, 21464–21480
1
1
1
1
1
6) 7.03 (t, J = 7.41 Hz, 1H, H-4 ), 7.29 (t, J = 7.72 Hz, 2H, H-3 , 5 ), 7.54 (d, J = 7.88 Hz, 2H, H-2 , 6 ),
9.67 (s, 1H, H-9), 11.99 (br, s, 1H, H-10); 13C-NMR δ: 28.91 (C-2), 29.46 (C-1,), 52.09 (C-4), 53.98 (C-5),
1
1
1
1
1
1
77.33 (C-3), 79.20 (C-6), 123.41 (C-2 , C-6 ), 127.28 (C-4 ), 129.06 (C-3 , C-5 ), 139.75 (C-1 ), 169.78 (C-8),
172.73 (C-7); HR-MS (ESI): m/z calcd. For C14H15NO4Na ([M + Na]+) 284.0899, found 284.0910.
3-(p-Tolylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-2). Yield 58%, white solid, mp:
˝
1
211–212 C; 1H-NMR δ: 1.44–1.70 (m, 4H, H-1, 2), 2.25 (s, 3H, H-7 ), 2.94 (d, J = 9.77 Hz, 1H, H-4), 3.05
(d, J = 9.46 Hz, 1H, H-5), 4.64 (d, J = 4.41 Hz, 1H, H-6), 4.79 (d, J = 3.78 Hz, 1H, H-3), 7.09 (d, J = 8.20 Hz
,
1
1
1
1
2H, H-3 , 5 ), 7.42 (d, J = 8.20 Hz, 2H, H-2 , 6 ) 9.56 (s, 1H, H-9) 11.95 (br, s, 1H, H-10); 13C-NMR δ:
1
20.90 (s, 1C, C-7 ), 28.90 (s, 1C, C-1), 29.46 (s, 1C, C-2), 52.07 (s, 1C, C-4), 53.97 (s, 1C, C-5), 77.31
1
1
1
1
1
(s, 1C, C-6), 79.22 (s, 1C, C-3), 119.68 (s, 2C, C-2 , C-6 ), 129.41 (s, 2C, C-3 , C5 ), 132.28 (s, 1C, C-1 ),
1
137.24 (s, 1C, C-4 ), 169.54 (s, 1C, C-8), 172.72 (s, 1C, C-7); HR-MS (ESI): m/z calcd for C15H17NO4Na
([M + Na]+) 298.1055, found 298.1051.
3-((2-Ethylphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-3). Yield 80%, white solid,
˝
1
mp: 172–173 C; 1H-NMR δ: 1.15 (t, J = 7.41 Hz, 3H, H-8 ), 1.54–1.73 (m, 4H, H-1, 2), 2.58 (q, J = 7.57 Hz
,
1
2H, H-7 ), 3.06–3.15 (m, 2H, H-4, 5), 4.74 (d, J = 5.04 Hz, 1H, H-6), 4.87 (d, J = 3.47 Hz, 1H, H-3),
1
1
1
1
7.05–7.10 (m, 1H, H-4 ), 7.13–7.22 (m, 2H, H-3 , 5 ), 7.66 (d, J = 7.88 Hz, 1H, H-6 ), 8.76 (s, 1H, H-9),
12.21 (br, s, 1H, H-10); 13C-NMR δ: 14.61 (C-8 ), 24.04 (C-7 ), 28.94 (C-1, C-2), 52.26 (C-4), 54.60
1
1
1
1
1
1
1
(C-5), 77.56 (C-3), 79.58 (C-6), 123.85 (C-6 ), 124.98 (C-3 ), 126.37 (C-5 ), 128.84 (C-4 ), 135.71 (C-2 ),
1
´
136.30 (C-1 ), 169.93 (C-8), 172.82 (C-7); HR-MS (ESI): m/z calcd for C16H18NO4 ([M ´ 1] ) 288.1236,
found 288.1246.
3-((2-Fluorophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-4). Yield 85%, white solid,
˝
mp: 196–197 C; 1H-NMR δ: 1.53–1.68 (m, 4H, H-1, 2), 3.04 (d, J = 9.46 Hz, 1H, H-4), 3.21 (d, J = 9.77 Hz
,
1
1
1H, H-5), 4.61–4.77 (m, 1H, H-6), 4.78–4.93 (m, 1H, H-3), 7.06–7.19 (m, 2H, H-3 , 5 ), 7.20–7.28 (m, 1H,
H-4 ), 8.02 (t, J = 7.72 Hz, 1H, H-6 ), 9.28 (s, 1H, H-9), 12.13 (br, s, 1H, H-10); 13C-NMR δ: 28.92 (s, 1C,
1
1
C-1), 29.11 (s, 1C, C-2), 52.28 (s, 1C, C-4), 54.13 (s, 1C, C-5), 77.54 (s, 1C, C-3), 79.36 (s, 1C, C-6), 115.53
1
1
1
1
1
(s, 1C, C-3 ), 123.24 (s, 1C, C-1 ), 124.83 (s, 1C, 6 ), 126.95 (s, 1C, 4 ), 152.24 (s, 1C, C-5 ), 154.17 (s, 1C,
1
C-2 ), 170.22 (s, 1C, C-8), 172.71 (s, 1C, C-7); HR-MS (ESI): m/z calcd. For C14H14FNO4Na ([M + Na]+)
302.0805, found 302.0807.
3-((3-Fluorophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-5). Yield 86%, white solid,
˝
mp: 165–167 C; 1H-NMR δ: 1.41–1.71 (m, 4H, H-1, 2), 2.97 (d, J = 9.46 Hz, 1H, H-4), 3.07 (d, J = 9.46 Hz
,
1H, H-5), 4.66 (d, J = 4.41 Hz, 1H, H-6), 4.80 (d, J = 3.78 Hz, 1H, H-3), 6.85 (td, J = 8.43, 2.36 Hz, 1H,
1
1
1
1
H-4 ), 7.22 (d, J = 8.20 Hz, 1H, H-6 ), 7.29–7.36 (m, 1H, H-5 ), 7.57 (d, J = 11.66 Hz, 1H, H-2 ), 9.95 (s,
1H, H-9) 12.01 (s, 1H, H-10); 13C-NMR δ: 28.89 (s, 1C, C-1), 29.44 (s, 1C, C-2), 52.17 (s, 1C, C-4), 53.89
1
1
(s, 1C, C-5), 77.38 (s, 1C, C-3), 79.07 (s, 1C, C-6), 106.28 (s, 1C, C-4 ), 109.72 (s, 1C, C-2 ), 115.31 (s, 1C,
1
1
1
1
C-6 ), 130.62 (s, 1C, C-5 ), 141.46 (s, 1C, C-1 ), 161.65 (s, 1C, C-3 ), 170.19 (s, 1C, C-8), 172.64 (s, 1C, C-7);
HR-MS (ESI): m/z calcd. For C14H14FNO4Na ([M + Na]+) 302.0805, found 302.0811.
3-((4-Fluorophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-6). Yield 78%, white solid,
˝
mp: 186–187 C; 1H-NMR δ: 1.45–1.69 (m, 4H, H-1, 2), 2.93–2.98 (m, 1H, H-4), 3.02–3.08 (m, 1H, H-5),
1
1
4.65 (d, J = 4.10 Hz, 1H, H-6), 4.79 (d, J = 3.78 Hz, 1H, H-3), 7.13 (t, J = 8.83 Hz, 2H, H-2 , 6 ), 7.55 (dd,
J = 8.83, 5.04 Hz, 2H, H-3 , 5 ), 9.73 (s, 1H, H-8), 11.98 (br, s, 1H, H-7); 13C-NMR δ: 28.90 (s, 1C, C-1),
1
1
29.46 (s, 1C, C-2), 52.15 (s, 1C, C-4), 53.83 (s, 1C, C-5), 77.34 (s, 1C, C-3), 79.10 (s, 2C, C-6), 115.49 (s, 2C,
1
1
1
1
1
1
C-3 , C-5 ), 121.37 (s, 2C, C-2 , C-6 ), 136.13 (s, 1C, C-1 ), 157.37 (s, 1C, C-4 ), 169.71 (s, 1C, C-8), 172.68
(s, 1C, C-7); HR-MS (ESI): m/z calcd. For C14H14FNO4Na ([M + Na]+) 302.0805, found 302.0813.
3-((2-Chlorophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-7). Yield 71%, white solid,
˝
1
mp: 177–178 C; H-NMR δ: 1.54–1.75 (m, 4H, H-1, 2), 3.15 (q, J = 9.77 Hz, 2H, H-4, 5), 4.78 (d,
1
J = 4.73 Hz, 1H, H-6), 4.91 (d, J = 3.47 Hz, 1H, H-3), 7.12 (t, J = 7.72 Hz, 1H, H-4 ), 7.32 (t, J = 7.72 Hz,
1H, H-5 ), 7.48 (d, J = 7.88 Hz, 1H, H-3 ), 8.05 (d, J = 7.88 Hz, 1H, H-6 ), 9.07 (s, 1 H, H-9), 12.30 (br, s,
1H, H-10); 13C-NMR δ: 28.80 (s, 1C, C-1), 28.97 (s, 1C, C-2), 52.43 (s, 1C, C-4), 54.87 (s, 1C, C-5), 77.68
1
1
1
21472

Molecules 2015, 20, 21464–21480
1
1
1
(s, 1C, C-3), 79.49 (s, 1C, C-6), 123.34 (s, 1C, C-6 ), 123.95 (s, 1C, C-5 ), 125.36 (s, 1C, C-2 ), 127.93 (s, 1C,
1
1
1
C-3 ), 129.64 (s, 1C, C-4 ), 135.54 (s, 1C, C-1 ), 170.25 (s, 1C, C-8), 172.61 (s, 1C, C-7); HR-MS (ESI): m/z
calcd. For C14H14ClNO4Na ([M + Na]+) 318.0509, found 318.0513.
3-((3-Chlorophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-8). Yield 55%, white solid,
˝
mp: 178–179 C; 1H-NMR δ: 1.44–1.70 (m, 4H, H-1, 2), 2.93–3.01 (m, 1H, H-4), 3.03-3.13 (m, 1H, H-5),
1
4.66 (d, J = 4.41 Hz, 1H, H-6), 4.79 (d, J = 3.78 Hz, 1H, H-3), 6.99–7.15 (m, 1H, H-4 ), 7.23–7.43 (m,
2H, H-5 , 6 ), 7.81 (s, 1 H, H-2 ), 9.93 (s, 1H, H-9), 12.01 (s, 1H, H-10); 13C-NMR δ: 28.89 (s, 1C, C-1),
1
1
1
29.44 (s, 1C, C-2), 52.20 (s, 1C, C-4), 53.84 (s, 1C, C-5), 77.38 (s, 1C, C-3), 79.03 (s, 1C, C-6), 117.97
1
1
1
1
1
(s, 1C, C-6 ), 119.17 (s, 1C, C-2 ), 123.09 (s, 1C, C-4 ), 130.75 (s, 1C, C-5 ), 133.44 (s, 1C, C-3 ), 141.18
1
(s, 1C, C-1 ), 170.21 (s, 1C, C-8), 172.63 (s, 1C, C-7); HR-MS (ESI): m/z calcd. For C14H14ClNO4Na
([M + Na]+) 318.0509, found 318.0515.
3-((4-Chlorophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-9). Yield 56%, white solid,
˝
mp: 178–181 C; 1H-NMR δ: 1.44–1.69 (m, 4H, H-1, 2), 2.97 (d, J = 9.46 Hz, 1H, H-4), 3.06 (d, J = 9.46 Hz
,
1
1
1H, H-5), 4.66 (d, J = 4.41 Hz, 1H, H-6), 4.79 (d, J = 3.47 Hz, 1H, H-3), 7.35 (d, J = 8.51 Hz, 2H, H-3 , 5 ),
7.57 (d, J = 8.83 Hz, 2H, H-2 , 6 ), 9.85 (s, 1H, H-8), 12.01 (s, 1H. H-7); 13C-NMR δ: 28.89 (s, 1C, C-1),
1
1
29.45 (s, 1C, C-2), 52.16 (s, 1C, C-4), 53.87 (s, 1C, C-5), 77.36 (s, 1C, C-3), 79.08 (s, 1C, C-6), 121.16 (s, 2C,
1
1
1
1
1
1
C-2 , C-6 ), 126.93 (s, 1C, C-4 ), 128.96 (s, 2C, C-3 , C-5 ), 138.73 (s, 1C, C-1 ), 169.96 (s, 1C, C-8), 172.66
(s, 1C, C-7); HR-MS (ESI): m/z calcd. For C14H14ClNO4Na ([M + Na]+) 318.0509, found 318.0519.
3-((2-Bromophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-10). Yield 45%, white solid,
˝
1
mp: 169–171 C; H-NMR δ: 1.55–1.73 (m, 4H, H-1, 2), 3.14 (s, 2H, H-4, 5), 4.79 (d, J = 4.73 Hz,
1
1
1H, H-6), 4.91 (d, J = 2.84 Hz, 1H, H-3), 7.06 (t, J = 7.57 Hz, 1H, H-4 ), 7.30–7.40 (m, 1H, H-5 ), 7.63 (d,
J = 7.88 Hz, 1H, H-3 ), 7.99 (d, J = 7.88 Hz, 1H, H-6 ), 8.99 (s, 1H, H-9 ), 12.29 (br, s, 1H, H-10); 13C-NMR
1
1
1
δ: 28.80 (s, 1C, C-1), 28.99 (s, 1C, C-2), 52.41 (s, 1C, C-4), 54.89 (s, 1C, C-5), 77.67 (s, 1C, C-3), 79.45 (s,
1
1
1
1
1C, C-6), 114.90 (s, 1C, C-2 ), 123.93 (s, 1C, C-6 ), 125.99 (s, 1C, C-5 ), 128.46 (s, 1C, C-3 ), 132.89 (s,
1
1
1C, C-4 ), 136.77 (s, 1C, C-1 ), 170.24 (s, 1C, C-8), 172.58 (s, 1C, C-7); HR-MS (ESI): m/z calcd. For
C14H14BrNO4Na ([M + Na]+) 362.0004, found 362.0008.
3-((3-Bromophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-11). Yield 56%, white solid,
˝
mp: 189–191 C; 1H-NMR δ: 1.45–1.70 (m, 4H, H-1, 2), 2.94–3.01 (m, 1H, H-4), 3.03–3.09 (m, 1H, H-5),
1
1
1
4.67 (br, s, 1H, H-6), 4.79 (br, s, 1H, H-3), 7.19–7.30 (m, 2H, H-5 , 6 ), 7.39 (d, J = 7.88 Hz, 1H, H-4 ),
7.96 (br, s, 1H, H-2 ), 9.91 (br, s, 1H, H-9), 12.01 (s, 1H, H-10); 13C-NMR δ: 28.90 (s, 1C, C-1), 29.44 (s,
1
1
1
1C, C-2), 52.21 (s, 1C, C-4), 53.84 (s, 1C, C-5), 77.40 (s, 1C, C-3), 79.03 (s, 1C, C-6), 118.36 (s, 1C, C-3 ),
1
1
1
1
121.94 (s, 1C, C-6 ), 122.05 (s, 1C, C-2 ), 125.99 (s, 1C, C-4 ), 131.06 (s, 1C, C-5 ), 141.32 (s, 1C, C-1 ),
170.19 (s, 1C, C-8), 172.63 (s, 1C, C-7); HR-MS (ESI): m/z calcd. For C14H14BrNO4Na ([M + Na]+)
362.0004, found 362.0011.
3-((4-Bromophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-12). Yield 65%, white solid,
˝
mp: 188–192 C; 1H-NMR δ: 1.44–1.69 (m, 4H, H-1, 2), 2.93–3.00 (m, 1H, H-4), 3.06 (d, J = 9.46 Hz, 1H,
1
1
H-5), 4.66 (d, J = 4.41 Hz, 1H, H-6), 4.79 (d, J = 3.78 Hz, 1H, H-3), 7.44–7.49 (m, 2H, H-3 , 5 ), 7.50-7.56
(m, 2H, H-2 , 6 ), 9.85 (s, 1H, H-9), 12.01 (s, 1H, H-10); 13C-NMR δ: 28.89 (s, 1C, C-1), 29.45 (s, 1C, C-2),
1
1
1
52.16 (s, 1C, C-4), 53.88 (s, 1C, C-5), 77.36 (s, 1C, C-3), 79.07 (s, 1C, C-6), 114.90 (s, 1C, C-4 ), 121.55
(s, 2C, C-2 , C-6 ), 131.87 (s, 2C, C-3 , C-5 ), 139.14 (s, 1C, C-1 ), 169.98 (s, 1C, C-8), 172.66 (s, 1C, C-7);
HR-MS (ESI): m/z calcd. For C14H14BrNO4Na ([M + Na]+) 362.0004, found 362.0015.
1
1
1
1
1
3-((2-Iodophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-13). Yield 74%, white solid,
˝
mp: 178–180 C; 1H-NMR δ: 1.54–1.75 (m, 4H, H-1, 2), 3.12 (br, s, 2H, H-3, 6), 4.81 (br, s, 1H, H-6), 4.91
1
1
(br, s, 1H, H-3), 6.91 (t, J = 7.09 Hz, 1H, H-4 ), 7.37 (t, J = 7.25 Hz, 1H, H-5 ), 7.77 (d, J = 7.57 Hz, 1H,
H-3 ), 7.85 (d, J = 7.57 Hz, 1H, H-6 ), 8.84 (br, s, 1H, H-9), 12.28 (br, s, 1H, H-10); 13C-NMR δ: 28.90
1
1
(1C, C-1), 29.02 (1C, C-2), 52.35 (1C, C-4), 54.66 (1C, C-5), 77.61 (1C, C-3), 79.33 (1C, C-6), 124.56 (1C,
1
1
1
1
1
C-2 ), 126.81 (1C, C-6 ), 128.98 (1C, C-4 ), 139.32 (1C, C-5 ), 139.74 (1C, C-3 ), 170.16 (1C, C-8), 172.58
(1C, C-7); HR-MS (ESI): m/z calcd. For C14H14INO4Na ([M + Na]+) 409.9865, found 409.9863.
21473

Molecules 2015, 20, 21464–21480
3-((3-Iodophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-14). Yield 54%, white solid,
˝
mp: 174–175 C; 1H-NMR δ: 1.41–1.71 (m, 4H, H-1, 2), 2.97 (d, J = 9.46 Hz, 1H, H-4), 3.07 (d, J = 9.46 Hz
,
1H, H-5), 4.66 (d, J = 4.41 Hz, 1H, H-6), 4.80 (d, J = 3.78 Hz, 1H, H-3), 6.85 (td, J = 8.43, 2.36 Hz, 1H,
1
1
1
1
H-4 ), 7.22 (d, J = 8.20 Hz, 1H, H-6 ), 7.29-7.36 (m, 1H, H-5 ), 7.57 (d, J = 11.66 Hz, 1H, H-2 ), 9.95 (s,
1H, H-9) 12.01 (s, 1H, H-10); 13C-NMR δ: 28.89 (s, 1C, C-1), 29.44 (s, 1C, C-2), 52.17 (s, 1C, C-4), 53.89
1
1
(s, 1C, C-5), 77.38 (s, 1C, C-3), 79.07 (s, 1C, C-6), 106.28 (s, 1C, C-4 ), 109.72 (s, 1C, C-2 ), 115.31 (s, 1C,
1
1
1
1
C-6 ), 130.62 (s, 1C, C-5 ), 141.46 (s, 1C, C-1 ), 161.65 (s, 1C, C-3 ), 170.19 (s, 1C, C-8), 172.64 (s, 1C, C-7);
HR-MS (ESI): m/z calcd. For C14H14INO4Na ([M + Na]+) 409.9865, 409.9871.
3-((3-Iodophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-15). Yield 81%, white solid,
˝
mp: 171–172 C; 1H-NMR δ: 1.47–1.69 (m, 4H, H-1, 2), 2.96 (d, J = 9.46 Hz, 1H, H-4), 3.05 (d, J = 9.46 Hz
,
,
1
1
1H, H-5), 4.65 (br, s, 1H, H-6), 4.79 (br, s, 1H, H-3), 7.39 (d, J = 8.51 Hz, 2H, H-3 , 5 ), 7.62 (d, J = 8.51 Hz
2H, H-2 , 6 ), 9.81 (br, s, 1H, H-9), 12.01 (br, s, 1H, H-10); 13C-NMR δ: 28.90 (1C, C-1), 29.44 (1C, C-2),
1
1
1
1
1
52.19 (1C, C-4), 53.93 (1C, C-5), 77.38 (1C, C-3), 79.06 (1C, C-6), 86.66 (1C, C-4 ), 121.86 (2C, C-2 , C-6 ),
1
1
1
137.69 (2C, C-3 , C-5 ), 139.61 (1C, C-1 ), 169.98 (1C, C-8), 172.65 (1C, C-7); HR-MS (ESI): m/z calcd.
For C14H14INO4Na ([M + Na]+) 409.9865, found 409.9871.
3-((4-Hydroxyphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-16). Yield 56%, white
˝
solid, mp: 174–175 C; 1H-NMR δ: 1.42–1.74 (m, 4H, H-1, 2) 2.89–2.96 (m, 1H, H-4) 3.02 (d, J = 9.46 Hz
,
1
1H, H-5) 4.62 (d, J = 3.78 Hz, 1H, H-6) 4.79 (d, J = 2.52 Hz, 1H, H-3) 6.68 (d, J = 8.20 Hz, 2H, H-3 ,
5 ) 7.30 (d, J = 8.51 Hz, 2H, H-2 , 6 ) 9.13 (br, s, 1H, H-7 ) 9.36 (s, 1H, H-9) 11.89 (br, s, 1H, H-10);
13C-NMR δ: 28.89 (s, 1C, C-1), 29.47 (s, 1C, C-2), 52.03 (s, 1C, C-4), 53.89 (s, 1C, C-5), 77.26 (s, 1C, C-3),
79.24 (s, 1C, C-6), 115.39 (s, 2C, C-3 , C-5 ), 121.49 (s, 2C, C-2 , C-6 ), 131.39 (s, 1C, C-1 ), 153.62 (s, 1C,
C-4 ), 169.13 (s, 1C, C-8), 172.77 (s, 1C, C-7); HR-MS (ESI): m/z calcd for C14H15NO5Na ([M + Na]+)
1
1
1
1
1
1
1
1
1
1
300.0848, found 300.0851.
3-((4-Carboxyphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-17). Yield 44%, white
˝
1
solid, mp: 256–257 C; H-NMR δ: 1.52–1.64 (m, 4H, H-1, 2), 2.98 (d, J = 9.46 Hz, 1H, H-4), 3.11 (d,
J = 9.77 Hz, 1H, H-5), 4.68 (d, J = 4.41 Hz, 1H, H-6), 4.80 (d, J = 3.78 Hz, 1H, H-3), 7.66 (d, J = 8.83 Hz
,
1
1
1
1
2H, H-2 , 6 ), 7.89 (d, J = 8.83 Hz, 2H, H-3 , 5 ), 10.05 (s, 1H, H-9), 12.35 (br, s, 2H, H-10); 13C-NMR
δ: 28.91 (s, 1C, C-1) 29.44 (s, 1C, C-2) 52.20 (s, 1C, C-4) 53.97 (s, 1C, C-5) 77.42 (s, 1C, C-3) 79.11 (s,
1
1
1
1
1
1
1C, C-6) 118.80 (s, 2C, C-2 , C-6 ) 125.29 (s, 1C, C-4 ) 130.78 (s, 2C, C-3 , C-5 ) 143.69 (m, 1C, C-1 )
1
167.32 (m, 1C, C-7 ) 170.32 (s, 1C, C-8) 172.65 (s, 1C, C-7); HR-MS (ESI): m/z calcd for C15H15NO6Na
([M + Na]+) 328.0797, found 328.0796.
3-((2-Methoxyphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-18). Yield 66%white
˝
1
solid, mp: 151–152 C; H-NMR δ: 1.52–1.72 (m, 4H, H-1, 2), 3.05–3.17 (m, 2H, H-4, 5), 3.83 (s,
1
1
1
1
3H, H-7 ), 4.71 (d, J = 5.04 Hz, 1H, H-6 ), 4.89 (d, J = 3.47 Hz, 1H, H-3 ), 6.86–6.93 (m, 1H, H-5 ),
1
1
1
7.02 (d, J = 4.10 Hz, 2H, H-3 , 4 ), 8.11 (d, J = 7.88 Hz, 1H, H-6 ), 8.87 (s, 1H, H-9), 12.20 (br, s, 1H, H-10);
13C-NMR δ: 28.96 (C-1, C-2), 52.44 (C-7 ), 55.32 (C-4), 56.45 (C-5), 77.52 (C-3), 79.54 (C-6), 111.44 (C-3 ),
1
1
1
1
1
1
1
120.32 (C-6 ), 120.79 (C-5 ), 123.84 (C-3 ), 128.31 (C-4 ), 148.84 (C-2 ), 169.95 (C-8), 172.61 (C-7); HR-MS
(ESI): m/z calcd for C15H17NO5Na ([M + Na]+) 314.1004, found 314.1008.
3-((3-Methoxyphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-19). Yield 73%, white
˝
1
solid, mp: 165–166 C; H-NMR δ: 1.47–1.70 (m, 4H, H-1, 2), 2.95 (d, J = 9.46 Hz, 1H, H-4), 3.07
(d, J = 9.77 Hz, 1H, H-5), 4.64 (d, J = 4.41 Hz, 1H, H-6), 4.80 (d, J = 3.78 Hz, 1H, H-3), 6.61 (dd, J = 8.20,
1
1
1
1
1.89 Hz, 1H, H-4 ), 7.05 (d, J = 8.20 Hz, 1H, H-6 ), 7.15–7.22 (m, 1 H, H-5 ), 7.29 (s, 1H, H-2 ), 9.68 (s, 1H,
1
H-9), 11.99 (br, s, 1H, H-10); 13C-NMR δ: 28.91 (C-2), 29.46 (C-1), 52.05 (C-7 ), 54.03 (C-4), 55.42 (C-5),
1
1
1
1
1
77.34 (C-3), 79.23 (C-6), 105.36 (C-6 ), 108.97 (C-2 ), 111.89 (C-4 ), 129.81 (C-5 ), 140.95 (C-1 ), 159.95
(C-3 ), 169.83 (C-8), 172.72 (C-7); HR-MS (ESI): m/z calcd for C15H17NO5Na ([M + Na]+) 314.1004,
1
found 314.0983.
3-((4-Methoxyphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-20). Yield 71%, white
˝
1
solid, mp: 167–168 C; H-NMR: 1.46–1.69 (m, 4H, H-1, 2), 2.94 (d, J = 9.77 Hz, 1H, H-4), 3.03 (d,
21474

Molecules 2015, 20, 21464–21480
J = 9.46 Hz, 1H, H-5), 4.63 (d, J = 4.10 Hz, 1H, 1H, H-6), 4.79 (d, J = 3.78 Hz, 1H, 1H, H-3), 6.87 (d,
1
1
1
1
J = 8.83 Hz, 2H, H-3 , 5 ), 7.44 (d, J = 8.83 Hz, 2 H, H-2 , 6 ), 9.51 (s, 1H, H-9), 11.93 (br, s, 1H, H-10);
1
13C-NMR δ: 28.89 (C-2), 29.47 (C-1), 52.07 (C-7 ), 53.87 (C-4), 55.64 (C-5), 77.29 (C-3), 79.20 (C-6),
1
1
1
1
1
1
114.20 (C-3 , C-5 ), 121.24 (C-2 , C-6 ), 132.91 (C-1 ), 155.54 (C-4 ), 169.33 (C-8), 172.76 (C-7); HR-MS
(ESI): m/z calcd for C15H17NO5Na ([M + Na]+) 314.1004, found 314.1025.
3-((4-(Trifluoromethoxy)phenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-21). Yield 47%,
˝
yellow solid, mp: 155–156 C; 1H-NMR δ: 1.46–1.69 (m, 4H, H-1, 2), 2.95–3.00 (m, 1H, H-4), 3.07 (d,
J = 9.77 Hz, 1H, H-5), 4.67 (d, J = 4.41 Hz, 1H, H-6), 4.80 (d, J = 3.78 Hz, 1H, H-3), 7.30 (d, J = 8.83 Hz,
1
1
1
1
2H, H-3 , 5 ), 7.65 (d, J = 8.83 Hz, 2H, H-2 , 6 ), 9.91 (s, 1H, H-9), 12.01 (s, 1H, H-10); 13C-NMR δ: 28.84
(m, 1C, C-1), 29.44 (m, 1C, C-2), 52.17 (m, 1C, C-4), 53.89 (m, 1C, C-5), 77.34 (m, 1C, C-3), 79.07 (m, 1C,
1
1
1
1
1
1
C-6), 120.88 (s, 2C, C-3 , C-5 ), 121.65 (s, 1C, C-7 ), 121.97 (s, 2C, C-2 , C-6 ), 138.91 (m, 1C, C-1 ), 143.80
1
(m, 1C, C-4 ), 169.85 (m, 1C, C-8), 172.47 (m, 1C, C-7); HR-MS (ESI): m/z calcd for C15H14F3NO5Na
([M + Na]+) 368.0722, found 368.0732.
3-((4-Cyanophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-22). Yield, 73%, yellow
˝
1
solid, mp: 164–166 C; H-NMR δ: 1.46–1.70 (m, 4H, H-1, 2), 3.00 (d, J = 9.46 Hz, 1H, H-4), 3.09
1
1
(d, J = 9.77 Hz, 1H, H-5), 4.69 (d, J = 2.52 Hz, 1H, H-6), 4.80 (br, s, 1H, H-3), 7.74 (s, 2H, H-3 , 5 ),
7.77–7.79 (m, 2H, H-2 , 6 ), 10.20 (s, 1H, H-9), 12.05 (s, 1H, H-10); 13C-NMR δ: 28.91 (s, 1C, C-1) 29.42
1
1
1
(s, 1C, C-2) 52.33 (s, 1C, C-4) 53.88 (m, 1C, C-5) 77.49 (s, 1C, C-3) 78.97 (s, 1C, C-6) 105.05 (s, 1C, C-4 )
119.56 (s, 1C, C-7 ) 133.65 (s, 2C, C-2 , C-6 ) 143.99 (s, 2C, C-3 , C-5 ) 170.62 (s, 1C, C-8) 172.57 (s, 1C,
C-7); HR-MS (ESI): m/z calcd for C15H14N2O4Na ([M + Na]+) 309.0851,found 309.0856.
1
1
1
1
1
3-((2-Nitrophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-23). Yield, 47%, yellow
˝
1
solid, mp: 155–156 C; H-NMR δ: 1.46–1.72 (m, 4H, H-1, 2), 2.98–3.12 (m, 2H, H-4, 5), 4.63–4.85
1
1
(m, 2H, H-3, 6), 7.60 (t, J = 8.20 Hz, 1H, H-4 ), 7.82 (d, J = 7.88 Hz, 1H, H-5 ), 7.90 (d, J = 7.88 Hz,
1H, H-6 ), 8.65 (br, s, 1H, H-3 ), 10.28 (br, s, 1H, H-9), 12.02-12.09 (m, 1H, H-10); 13C-NMR δ: 28.91
1
1
1
1
(C-2), 29.44 (C-1), 52.24 (C-4), 53.82 (C-5), 77.49 (C-3), 79.00 (C-6), 113.68 (C-6 ), 117.96 (C-4 ), 125.55
1
1
1
1
(C-3 ), 130.53 (C-5 ), 140.91 (C-1 ), 148.42 (C-2 ), 170.61 (C-8), 172.63 (C-7); HR-MS (ESI): m/z calcd for
C14H14N2O6Na ([M + Na]+) 329.0750, found 329.0754.
3-((3-Nitrophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-24). Yield, 44%, yellow
˝
1
solid, mp: 178–179 C; H-NMR δ: 1.45–1.70 (m, 4H, H-1, 2), 2.94–3.01 (m, 1H, H-4), 3.03–3.09 (m,
1
1
1H, H-5), 4.67 (br, s, 1H, H-6), 4.79 (br, s, 1H, H-3), 7.19–7.30 (m, 2H, H-4 , 6 ), 7.39 (d, J = 7.88 Hz,
1H, H-5 ), 7.96 (br, s, 1H, H-2 ), 9.91 (br, s, 1H, H-9) 12.01 (s, 1H, H-10); 13C-NMR δ: 28.54 (C-2), 29.36
(C-1), 52.44 (C-4), 53.79 (C-5), 77.80 (C-3), 79.55 (C-6), 115.24 (C-2 ), 119.30 (C-4 ), 126.72 (C-6 ), 130.81
(C-5 ), 140.68 (C-1 ), 149.11 (C-3 ); HR-MS (ESI): m/z calcd for C14H14N2O6Na ([M + Na]+) 329.0750,
1
1
1
1
1
1
1
1
found 329.0771.
3-((4-Nitrophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-25). Yield, 54%, yellow
˝
1
solid, mp: 156–157 C; H-NMR δ: 1.48–1.70 (m, 4H, H-1, 2), 3.02 (d, J = 9.46 Hz, 1H, H-4), 3.12
1
1
(d, J = 9.46 Hz, 1H, H-5), 4.71 (br, s, 1H, H-6), 4.81 (br, s, 1H, H-3), 7.80 (d, J = 8.83 Hz, 2H, H-2 , 6 ),
8.22 (d, J = 8.83 Hz, 2H, H-3 , 5 ), 10.40 (s, 1H, H-9), 12.09 (br, s, 1H, H-10); 13C-NMR δ: 28.92 (C-2),
1
1
1
1
1
1
29.42 (C-1), 52.39 (C-4), 53.89 (C-5), 77.56 (C-3), 78.96 (C-6), 119.15 (C-2 , C-6 ), 125.40 (C-3 , C-5 ),
1
1
142.41 (C-4 ), 146.01 (C-1 ), 170.79 (C-8), 172.56 (C-7); HR-MS (ESI): m/z calcd for C14H14N2O6Na
([M + Na]+) 329.0750, found 329.0763.
3-((3,4-Dichlorophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-26). Yield, 88%, white
˝
solid, mp: 160–161 C; 1H-NMR δ: 1.45–1.69 (m, 4H, H-1, 2), 2.95–3.01 (m, 1H, H-4), 3.02–3.08 (m, 1H,
1
H-5), 4.67 (d, J = 3.78 Hz, 1H, H-6), 4.79 (d, J = 2.84 Hz, 1H, H-3), 7.40 (d, J = 8.83 Hz, 1H, H-5 ), 7.55
(d, J = 8.83 Hz, 1H, H-6 ), 7.99 (s, 1H, H-2 ), 10.06 (s, 1H, H-9), 12.04 (s, 1H, H-10); 13C-NMR δ: 28.86
(m, 1C), 29.43 (s, 1C), 52.26 (s, 1C), 53.78 (s, 1C), 77.42 (s, 1C), 78.95 (s, 1C), 119.64 (s, 1C), 120.86 (s,
1C), 124.77 (s, 1C), 131.02 (s, 1C), 131.32 (s, 1C), 139.80 (s, 1C), 170.29 (s, 1C), 172.61 (s, 1C); HR-MS
(ESI): m/z calcd for C14H13Cl2NO4Na ([M + Na]+) 352.0119, found 352.0113.
1
1
21475

Molecules 2015, 20, 21464–21480
3-((2,4-Dibromophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-27). Yield, 18%, white
˝
solid, mp: 241–242 C; 1H-NMR δ: 1.59–1.68 (m, 4H, H-1, 2), 3.14 (s, 2H, H-4, 5), 4.79 (d, J = 4.10 Hz,
1
1
1H, H-6), 4.91 (br, s, 1H, H-3), 7.58 (d, J = 8.83 Hz, 1H, H-5 ), 7.88 (s, 1H, H-6 ), 7.95 (d, J = 8.83 Hz, 1H,
H-3 ), 9.06 (s, 1H, H-9), 12.31 (br, s, 1H, H-10); 13C-NMR δ: 28.77 (1C, C-1), 28.99 (1C, C-2), 52.46 (1C,
1
1
1
C-4), 54.78 (1C, C-5), 77.72 (1C, C-3), 79.37 (1C, C-6), 115.70 (1C, C-4 ), 116.51 (1C, C-2 ), 125.09 (1C,
C-6 ), 131.40 (1C, C-3 ), 134.66 (1C, C-5 ), 136.36 (1C, C-1 ), 170.37 (1C, C-8), 172.56 (1C, C-7); HR-MS
(ESI): m/z calcd for C14H13Br2NO4Na ([M + Na]+) 441.9089, found 441.9096.
1
1
1
1
3-((4-Iodo-2-methylphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-28). Yield, 56%,
˝
1
1
white solid, mp: 147–149 C; H-NMR δ: 1.59 (br, s, 4H, H-1, 2), 2.26 (s, 3H, H-7 ), 3.04–3.10 (m,
1
1H, H-4), 3.12–3.18 (m, 1H, H-5), 4.74–4.79 (m, 1H, H-6), 4.81–4.87 (m, 1H, H-3), 7.42 (s, 1H, H-5 ),
1
1
7.67 (d, J = 8.20 Hz, 1H, H-6 ), 7.80 (d, J = 7.88 Hz, 1H, H-4 ), 9.29–9.35 (m, 1H, H-9), 12.17–12.21 (m,
1H, H-10); 13C-NMR δ: 13.76 (1C, C-7 ), 28.91 (1C, C-1), 29.22 (1C, C-2), 52.33 (1C, C-4), 53.72 (1C,
1
1
1
1
C-5), 77.57 (1C, C-3), 79.13 (1C, C-6), 120.49 (1C, C-4 ), 125.27 (1C, C-6 ), 126.95 (1C, C-2 ), 129.20 (1C,
1
1
1
C-5 ), 128.61 (1C, C-1 ), 151.26 (1C, C-3 ), 170.18 (1C, C-8), 172.59 (1C,C-7); HR-MS (ESI): m/z calcd for
C15H16INO4Na ([M + Na]+) 424.0022, found 424.0025.
3-((3,5-Dimethoxyphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-29). Yield, 74%, white
˝
1
solid, mp: 156–157 C; H-NMR δ: 1.47–1.68 (m, 4H, H-1, 2), 2.94 (d, J = 9.77 Hz, 1H, H-4), 3.05 (d,
1
1
J = 9.46 Hz, 1H, H-5), 3.71 (s, 6H, H-7 , 8 ), 4.63 (d, J = 3.47 Hz, 1H, H-6), 4.79 (br, s, 1H, H-3), 6.20
(br, s, 1H, H-4 ), 6.80 (s, 2H, H-2 , 6 ), 9.67 (s, 1H, H-9), 11.98 (br, s, 1H, H-10); 13C-NMR δ: 28.91 (1C,
1
1
1
1
1
C-1), 29.42 (1C, C-2), 40.02 (2C, C-7 , C-8 ), 52.33 (1C, C-4), 53.89 (1C, C-5), 77.49 (1C, C-3), 78.97 (1C,
1
1
1
1
1
1
C-6), 105.05 (1C, C-4 ), 119.56 (2C, C-2 , C-6 ), 133.65 (1C, C-1 ), 143.99 (2C, C-3 , C-5 ), 170.62 (1C, C-8),
172.57 (1C, C-7); HR-MS (ESI): m/z calcd for C16H19NO6Na ([M + Na]+) 344.1110, found 344.1111.
3-((2,5-Dimethoxyphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-30). Yield 64%, white
˝
solid, mp: 188–189 C; 1H-NMR δ: 1.58 (br, s, 4H, H-1, 2), 2.97–3.23 (m, 2H, H-4, 5), 3.55–3.94 (m, 6H,
1
1
1
1
H-7 , 8 ), 4.71 (br, s, 1H, H-6), 4.89 (br, s, 1H, H-3), 6.58 (br, s, 1H, H-4 ), 6.94 (br, s, 1H, H-3 ), 7.83 (br, s,
1H, H-6 ), 8.89 (br, s, 1H, H-9), 12.10–12.40 (m, 1H, H-10); 13C-NMR δ: 28.8 (1C, C-1), 28.96 (1C, C-2),
52.46 (1C, C-4), 55.33 (1C, C-5), 55.80 (1C, C-8 ), 57.11 (1C, C-7 ), 77.57 (1C, C-3), 79.50 (1C, C-6), 107.14
(1C, C-6 ), 107.58 (1C, C-3 ), 112.29 (1C, C-4 ), 129.20 (1C, C-1 ), 143.25 (1C, C-2 ), 153.64 (1C, C-5 ),
170.11 (1C, C-8), 172.59 (1C, C-7); HR-MS (ESI): m/z calcd for C16H19NO6Na ([M + Na]+) 344.1110,
found 344.1119.
1
1
1
1
1
1
1
1
1
3-((2-Methyl-3-nitrophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-31). Yield, 35%,
˝
1
1
white solid, mp: 144–145 C; H-NMR δ: 1.59 (br, s, 4H, H-1, 2), 2.26 (s, 3H, H-7 ), 3.04–3.10 (m,
1
1H, H-4), 3.12–3.18 (m, 1H, H-5), 4.74–4.79 (m, 1H, H-6), 4.81–4.87 (m, 1H, H-3), 7.42 (s, 1H, H-5 ),
1
1
7.67 (d, J = 8.20 Hz, 1H, H-6 ), 7.80 (d, J = 7.88 Hz, 1H, H-4 ), 9.29–9.35 (m, 1H, H-9), 12.17–12.21 (m,
1H, H-10); 13C-NMR δ: 13.76 (1C, C-7 ), 28.91 (1C, C-1), 29.22 (1C, C-2), 52.33 (1C, C-4), 53.72 (1C,
1
1
1
1
C-5), 77.57 (1C, C-3), 79.13 (1C, C-6), 120.49 (1C, C-4 ), 125.27 (1C, C-6 ), 126.95 (1C, C-2 ), 129.20 (1C,
1
1
1
C-5 ), 128.61 (1C, C-1 ), 151.26 (1C, C-3 ), 170.18 (1C, C-8), 172.59 (1C,C-7); HR-MS (ESI): m/z calcd for
C15H16N2O6Na ([M + Na]+) 343.0906, found 343.0911.
3-(Naphthalen-1-ylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-32). Yield, 69%, white solid,
˝
mp: 187–188 C; 1H-NMR δ: 1.62 (br, s, 4H, H-1, 2), 3.11 (d, J = 9.77 Hz, 1H, H-4), 3.29 (s, 1H, H-5),
1
1
1
4.79-4.85 (m, 1H, H-6), 4.86–4.91 (m, 1H, H-3), 7.49 (s, 1H, H-2 ), 7.53–7.61 (m, 2H, H-3 , 9 ), 7.73 (s,
1H, H-8 ), 7.78–7.84 (m, 1H, H-4 ), 7.91–7.96 (m, 1H, H-7 ), 8.03–8.08 (m, 1H, H-10 ), 9.57 (s, 1H, H-9),
12.18 (s, 1H, H-10); 13C-NMR δ: 28.97 (1C, C-1), 29.28 (1C, C-2), 52.36 (1C, C-4), 54.14 (1C, C-5), 77.60
1
1
1
1
1
1
1
1
(1C, C-3), 79.49 (1C, C-6), 120.54 (1C, C-2 ), 122.63 (1C, C-4 ), 125.01 (1C, C-7 ), 125.99 (1C, C-6 ), 126.28
1
1
1
1
1
1
(1C, C-8 ), 126.43 (1C, C-9 ), 127.47 (1C, C-3 ), 128.61 (1C, C-10 ), 134.04 (1C, C-5 ), 134.09 (1C, C-1 ),
170.35 (1C, C-8), 172.86 (1C, C-7); HR-MS (ESI): m/z calcd for C18H17NO4Na ([M + Na]+) 334.1055,
found 334.1056.
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Molecules 2015, 20, 21464–21480
3-(Pyridin-2-ylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-33). Yield, 46%, white solid,
˝
mp: 178–180 C; 1H-NMR δ: 1.46–1.68 (m, 4H, H-1, 2), 3.02 (d, J = 9.46 Hz, 1H, H-4), 3.21 (d, J = 9.46 Hz
,
1
1H, H-5), 4.69 (d, J = 5.04 Hz, 1H, H-6), 4.83 (d, J = 3.78 Hz, 1H, H-3), 7.33–7.40 (m, 1H, H-4 ), 7.76
(t, J = 7.72 Hz, 1H, H-5 ), 7.89 (d, J = 4.41 Hz, 1H, H-6 ), 8.03 (d, J = 8.20 Hz, 1H, H-3 ), 9.97 (s, 1H,
H-9); 13C-NMR δ: 28.90 (1C, C-1), 29.24 (1C, C-2), 52.28 (1C, C-4), 53.92 (1C, C-5), 77.54 (1C, C-3),
79.28 (1C, C-6), 119.65 (1C, C-6 ), 137.48 (1C, C-4 ), 147.97 (1C, C-5 ), 152.43 (1C, C-3 ), 160.07 (1C,
C-1 ), 170.60 (1C, C-8), 172.74 (1C, C-7); HR-MS (ESI): m/z calcd for C13H15N2O4 ([M + 1] +) 263.1135,
1
1
1
1
1
1
1
1
found 263.1033.
3-(Thiazol-2-ylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-34). Yield, 56%, white solid, mp:
˝
174–176 C; 1H-NMR δ: 1.42–1.70 (m, 4H, H-1, 2), 3.02 (d, J = 9.46 Hz, 1H, H-4), 3.22 (d, J = 9.46 Hz,
1
1H, H-5), 4.67 (d, J = 4.10 Hz, 1H, H-6), 4.80 (d, J = 3.15 Hz, 1H, H-3), 7.18 (d, J = 3.47 Hz, 1H, H-5 ),
7.45 (d, J = 3.15 Hz, 1H, H-4), 11.83–11.98 (m, 1H, H-9), 12.00–12.18 (m, 1H, H-10); 13C-NMR δ: 28.85
(1C, C-1), 29.42 (1C, C-2), 52.30 (1C, C-4), 52.43 (1C, C-5), 77.61 (1C, C-3), 79.05 (1C, C-6), 113.62 (1C,
1
1
1
C-5 ), 137.92 (1C, C-4 ), 158.54 (1C, C-1 ), 169.81 (1C, C-8), 172.45 (1C, C-7); HR-MS (ESI): m/z calcd
for C11H12N2O4SNa ([M + Na]+) 291.0415, found 291.0411.
3-(Benzo[d]thiazol-2-ylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-35). Yield, 89%, white
˝
solid, mp: 175–176 C; 1H-NMR: 1.45–1.73 (m, 4H, H-1, 2), 3.09 (d, J = 9.14 Hz, 1H, H-4), 3.24 (br, s,
1
1
1
1H, H-5), 4.75 (br, s, 1H, H-6), 4.82 (br, s, 1H, H-3), 7.26–7.47 (m, 2H, H-7 , 8 ), 7.70–7.77 (m, 1H, H-6 ),
7.94-8.02 (m, 1H, H-9 ), 12.19 (br, s, 2H, H-9, 10); 13C-NMR δ: 28.87 (1C, C-1), 29.38 (1C, C-2), 52.55
(1C, C-4), 52.63 (1C, C-5), 77.84 (1C, C-3), 78.86 (1C, C-6), 120.84 (1C, C-9 ), 122.07 (1C, C-6 ), 123.77
(1C, C-7 ), 126.46 (1C, C-8 ), 131.84 (1C, C-3 ), 149.01 (1C, C-4 ), 158.52 (1C, C-1 ), 170.82 (1C, C-8),
172.31 (1C, C-7); HR-MS (ESI): m/z calcd for C15H14N2O4SNa ([M + Na]+) 341.0572, found 341.0579.
1
1
1
1
1
1
1
1
3-((1H-Benzo[d]imidazol-2-yl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-36). Yield, 45%,
˝
1
white solid, mp: 189–190 C; H-NMR δ: 1.42–1.70 (m, 4H, H-1, 2), 3.02 (d, J = 9.46 Hz, 1H, H-4),
3.22 (d, J = 9.46 Hz, 1H, H-5), 4.67 (d, J = 4.10 Hz, 1H, H-6), 4.80 (d, J = 3.15 Hz, 1H, H-3), 7.28 (dd,
1
1
1
1
J = 5.36, 2.84 Hz, 2H, H-6 , 9 ), 7.61 (br, s, 2H, H-7 , 8 ), 10.15 (s, 1H, H-9), 12.05 (s, 1H, H-10); 13C-NMR
δ: 28.90 (1C, C-1), 29.47 (1C, C-2), 52.11 (1C, C-4), 53.88 (1C, C-5), 77.33 (1C, C-3), 79.20 (1C, C-6),
1
1
1
1
1
1
1
111.90 (2C, C-6 , C-9 ), 119.73 (2C, C-7 , C-8 ), 138.96 (2C, C-3 , C-4 ), 155.36 (1C, C-1 ), 176.17 (2C, C-7,
C-8); HR-MS (ESI): m/z calcd for C15H15N3O4Na ([M + Na]+) 324.0960, found 324.0963.
3.3. Screening of Antifungal Activity in Vitro
The antifungal activity of the synthetic compounds in vitro against eight plant pathogenic fungi
(Valsa mali, B. berengeriana, S. fructigena, G. cingulate, A. alternate, S. sclerotiorum, A. solani and C.
sativum) was assayed by the mycelium growth rate method with slight modification [27]. All of the
fungi were provided by Laboratory of Integrated Management of Plant Diseases, Northwest A & F
˝
University (Yangling, China). The isolates were cultured for 5 days at 25 ˘ 1 C on potato dextrose
agar (PDA).
Antifungal activity was assessed as follows: the synthesized compounds were screened in vitro
for their antifungal activities against the eight phytopathogenic fungi. PDA medium was prepared in
the flasks and sterilized. Those compounds were dissolved in DMSO at a concentration of 50 µg/mL.
DMSO served as the control, while commercially available agricultural fungicide thiabendazole was
used as a positive control for its high efficiency and broad spectrum of antifungal activity. Each
sample was measured in three replicates, each colony diameter of the three replicates was measured
four times by a cross bracketing method. After the mycelia completed growth, the diameters of the
mycelial masses were measured and the inhibition rates were calculated according to the following
formula and expressed as means ˘ S.D.:
Growth inhibition rate p%q “ rpdc ´ d0q ´ pdt ´ d0qs{pdc ´ d0q ˆ 100
(1)
21477

Molecules 2015, 20, 21464–21480
where d0: diameter of the fungus cut-outs, dc: average diameter of the untreated control fungus, and
dt is the average diameter of mycelia on treated PDA with those compounds.
Based on the results of preliminary screening, the final stock solutions of the tested compounds
dissolved in acetone were 100, 50, 25, 10, and 5 µg/mL. The medium was then poured into sterilized
˝
Petri dishes. All types of fungi were incubated in PDA at 25 ˘ 1 C for 5 days to get new mycelium
for the antifungal assays, and a mycelia disk of approximately 5 mm diameter cut-out from culture
medium was picked up with a sterilized inoculation needle and inoculated in the center of the PDA
Petri dishes with different concentrations of NCTD derivatives [29,30]. The inoculated Petri dishes
˝
were incubated at 25 ˘ 1 C for 4 days. The IC50 (median inhibitory concentration) values of some
compounds were determined, and the results are listed in Table 2.
4. Conclusions
In summary, we have reported the synthesis of a series of NCTD derivatives with aromatic
amine moieties as well as the ability of these compounds to inhibit the growth of eight fungal
phytopathogens. Seven of these synthetic compounds presented significant fungistatic activities
against all of the eight fungi, superior to the corresponding parent compound NCTD for some fungi,
and in some cases they were the same as or more active than TBZ. Compound II-8 exhibited the
most significant activity on all eight fungi, much better than TBZ, NCTD and CTD. In particular,
II-8 showed excellent antifungal properties against S. fructigena and S. sclerotiorum, with IC50 values
of 0.88 and 0.97 µg/mL, respectively. SAR data for these compounds are as follows: (1) the
benzene ring is critical for the improvement of the spectrum of antifungal activity and the inhibition
of B. berengeriana, G. cingulate, A. alternate, S. sclerotiorum, A. solani and C. sativum (c.f. II-1 vs.
1
1
1
norcantharidin and cantharidin); (2) among the three sites, including the C-2 , C-3 and C-4 positions
1
of the phenyl ring, the presence of the halogen atom at the C-3 position of the phenyl ring caused
the most significant increase in antifungal activity (II-5 vs. II-4 and II-6, II-8 vs. II-7 and II-9, II-11 vs.
II-10 and II-12); (3) compounds with substitutions of strongly electron-drawing or electron-donating
groups were found to have a poor antifungal activity; and (4) compared with fluorine, bromine and
1
iodine, one chlorine atom substituted at C-3 position of the benzene ring gave the highest fungistatic
activity (II-8 vs. II-5, II-11 and II-14). Taken together, the data demonstrated that compound II-8
possesses the most potent inhibitory activity toward the fungal plant pathogens tested in this study
and could be a potential lead structure for further discovery of novel antifungal agrochemicals.
Acknowledgments: We sincerely appreciate John Richard Schrock (Emporia State University, Kansas, USA) and
Sang-Youl Park (University of California, Riverside, CA, USA) for revising the manuscript. This research is
supported by the Special Fund for the Public Interest (Agriculture) (200903052) by The Ministry of Science and
Technology and The Ministry of Agriculture of China, the Shaanxi Agriculture Science and Technology Projects
(2014K01-25-02) and The Ministry of Agriculture of China and the “13115” Sci-Tech Innovation Project of Shaanxi
Province (2007ZDKG-14).
Author Contributions: W.S. and Y.W. conceived and designed the experiments; W.S., S.Z. and H.W. performed
the experiments; W.S. and S.Z. analyzed the data; Y.Z. contributed reagents/materials/analysis tools; W.S. and
Y.W. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open
access article distributed under the terms and conditions of the Creative Commons by
Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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