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. 2015 Dec 2;20(12):21464–21480. doi: 10.3390/molecules201219782

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, Yalin Zhang 2,*
Editor: Derek J McPhee
PMCID: PMC6331962  PMID: 26633341

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, 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.

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 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 [11,12,13,14], CTD also shows interesting antifungal and insecticidal activities [15,16,17,18].

Figure 1.

Figure 1

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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.

Figure 2

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Design of the title compounds.

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.

Scheme 1.

Scheme 1

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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.

Table 1.

Preliminary antifungal activities of compounds at 50 μg/mL.

Compd. Values of Inhibition Rate (%) to Eight Pathogens
Valsa mali Botryosphaeria berengeriana Sclerotinia fructigena Glomerella cingulate Alternaria alternate Sclerotinia sclerotiorum Alternaria solani Cochliobolus sativum
II-1 68.42 ± 1.99d * 68.64 ± 0.65e 72.20 ± 0.66e 62.35 ± 0.99f 65.52 ± 1.06de 76.39 ± 0.92e 61.10 ± 0.96e 67.01 ± 0.59c
II-2 0.66 ± 0.06q 1.27 ± 0.03qr 0.53 ± 0.06qr 5.38 ± 0.05tu 9.66 ± 0.06no 23.61 ± 0.06p 1.52 ± 0.10u 7.98 ± 0.06qr
II-3 18.42 ± 0.06mn 11.02 ± 0.10l 18.72 ± 0.06m 6.99 ± 0.06st 11.72 ± 0.06n 20.17 ± 0.00q 1.01 ± 0.06u 10.11 ± 0.06pqr
II-4 74.99 ± 1.30c 69.90 ± 1.17e 76.48 ± 1.63d 80.66 ± 1.18d 70.34 ± 1.41c 86.69 ± 0.85d 76.77 ± 0.26c 66.50 ± 0.30c
II-5 83.56 ± 1.05b 87.70 ± 0.91c 85.03 ± 1.79bc 90.34 ± 2.64b 84.82 ± 1.30a 92.27 ± 0.06c 83.35 ± 1.16b 81.39 ± 0.38b
II-6 41.45 ± 0.47e 39.39 ± 1.61f 49.21 ± 1.37f 55.40 ± 1.36g 41.38 ± 2.14g 46.34 ± 2.29f 47.46 ± 1.21f 51.59 ± 0.76e
II-7 34.21 ± 1.00f 22.86 ± 1.85h 45.47 ± 2.26g 24.74 ± 0.83h 38.62 ± 1.04g 35.19 ± 1.78g 30.29 ± 0.92g 35.06 ± 1.80f
II-8 86.85 ± 1.00a 91.54 ± 1.33b 100.00 ± 0.00a 92.96 ± 2.69a 86.90 ± 2.32a 100.00 ± 0.00a 89.90 ± 0.79a 91.50 ± 0.66a
II-9 32.88 ± 3.85f 22.01 ± 2.89h 28.36 ± 2.69h 24.18 ± 1.08h 30.34 ± 3.13h 26.60 ± 2.79i 28.27 ± 1.83hi 17.53 ± 0.90j
II-10 25.01 ± 3.06g 18.18 ± 3.79i 23.00 ± 2.51i 21.48 ± 1.28i 23.44 ± 2.27i 30.46 ± 3.04h 18.17 ± 2.45j 21.21 ± 2.53i
II-11 80.92 ± 1.26b 81.77 ± 0.90d 83.42 ± 2.49c 84.97 ± 1.47c 78.63 ± 2.28b 96.13 ± 1.32b 79.83 ± 1.81bc 83.56 ± 1.80b
II-12 20.39 ± 1.04h 15.24 ± 3.23i 17.13 ± 2.59j 18.25 ± 1.37j 22.07 ± 3.14i 20.59 ± 2.45j 25.23 ± 1.87i 13.78 ± 1.91k
II-13 15.14 ± 1.23i 10.99 ± 2.47j 22.46 ± 1.62i 12.35 ± 0.59k 17.22 ± 4.17j 15.87 ± 1.37k 16.16 ± 2.31j 9.54 ± 1.22l
II-14 65.79 ± 1.00d 66.51 ± 1.95e 70.60 ± 2.19e 60.77 ± 0.78f 62.76 ± 2.13e 73.80 ± 3.43e 58.60 ± 1.33e 63.86 ± 1.01d
II-15 3.29 ± 1.16j −5.12 ± 3.86k 1.07 ± 0.92k 5.37 ± 0.86l 3.44 ± 1.18k 8.15 ± 1.43l 11.10 ± 6.18k −2.71 ± 1.98m
II-16 9.21 ± 0.10p 0.85 ± 0.04pqr 8.56 ± 0.06p 9.14 ± 0.03rs 10.34 ± 0.06no 6.87 ± 0.06t 2.02 ± 0.10u 5.85 ± 0.10s
II-17 0.66 ± 0.06q 0.42 ± 0.06qr −0.53 ± 0.06r 0.54 ± 0.06wx 1.38 ± 0.06pq 0.43 ± 0.08u 7.58 ± 0.12s 12.23 ± 0.10nopq
II-18 1.97 ± 0.06q 6.36 ± 0.06mn 0.00 ± 0.00qr 0.00 ± 0.00x 8.28 ± 0.06no 0.00 ± 0.06u 0.00 ± 0.00u 0.00 ± 0.00t
II-19 0.66 ± 0.06q 10.17 ± 0.06l 2.67 ± 0.06qr 11.83 ± 0.05pq 15.86 ± 0.06m 0.00 ± 0.06u 2.53 ± 0.00u 11.70 ± 0.06opq
II-20 0.66 ± 0.06q 2.12 ± 0.10pqr 0.53 ± 0.06qr 0.00 ± 0.00x 2.07 ± 0.06pq 0.00 ± 0.06u 0.00 ± 0.00u 0.00 ± 0.00t
II-21 1.32 ± 0.10q 10.59 ± 0.06l 11.76 ± 0.10o 15.59 ± 0.06o 20.69 ± 0.06ijk 13.30 ± 0.06s 21.72 ± 0.06lm 22.87 ± 0.06hi
II-22 24.34 ± 0.06k 0.00 ± 0.066r 8.02 ± 0.11p 12.90 ± 0.05p 17.93 ± 0.06jklm 18.45 ± 0.06q 22.73 ± 0.06kl 12.23 ± 0.03nopq
II-23 19.74 ± 0.06m 0.00 ± 0.00r 9.09 ± 0.11p 3.23 ± 0.03uv 0.69 ± 0.10pq 13.73 ± 0.10t 2.53 ± 0.03u 11.17 ± 0.06opq
II-24 25.00 ± 0.10q 5.08 ± 0.06n 18.18 ± 0.10m 20.43 ± 0.06m 28.28 ± 0.06h 19.31 ± 0.06q 7.07 ± 0.03st 15.43 ± 0.10lm
II-25 23.03 ± 0.10kl 2.54 ± 0.11pq 12.30 ± 0.06o 17.20 ± 0.05no 21.38 ± 0.10ij 7.73 ± 0.06u 3.54 ± 0.03tu 6.38 ± 0.11rs
II-26 32.89 ± 0.10j 18.22 ± 0.06k 48.13 ± 0.06g 28.49 ± 0.06h 35.86 ± 0.10g 40.34 ± 0.06k 33.33 ± 0.12op 37.23 ± 0.06f
II-27 36.18 ± 0.06hi 15.25 ± 0.06j 39.57 ± 0.06i 22.04 ± 0.06jkl 20.69 ± 0.06ijk 42.96 ± 0.06j 29.80 ± 0.06ij 14.89 ± 0.06lmn
II-28 8.55 ± 0.06p 6.78 ± 0.06o 39.04 ± 0.06i 23.66 ± 0.06ijk 38.62 ± 0.10fg 42.06 ± 0.00jk 16.67 ± 0.10nop 12.77 ± 0.06mnop
II-29 30.26 ± 0.06j 0.42 ± 0.06qr 36.36 ± 0.06j 21.51 ± 0.06kl 40.00 ± 0.06f 36.48 ± 0.06l 20.20 ± 0.06lmn 12.77 ± 0.10mnop
II-30 0.66 ± 0.06q 0.00 ± 0.06r 14.97 ± 0.10n 4.30 ± 0.06tuv 10.34 ± 0.16no 26.18 ± 0.06no 12.12 ± 0.10qr 18.62 ± 0.10jk
II-31 15.13 ± 0.10o 0.00 ± 0.06r 17.11 ± 0.06m 4.84 ± 0.00tuv -1.38 ± 0.06q 12.02 ± 0.06s 7.07 ± 0.10st 12.23 ± 0.11nopq
II-32 3.29 ± 0.10 q 8.05 ± 0.06m 6.42 ± 0.06p 5.91 ± 0.06tu 6.90 ± 0.06o 7.30 ± 0.00t 18.69 ± 0.06mno 22.34 ± 0.06hi
II-33 0.66 ± 0.06q 0.00 ± 0.11r 28.88 ± 0.06k 2.69 ± 0.06vw -0.69 ± 0.05q 23.61 ± 0.08p 6.57 ± 0.06st −1.60 ± 0.07t
II-34 36.84 ± 0.10h 0.42 ± 0.06qr 30.48 ± 0.06k 0.54 ± 0.06x 0.69 ± 0.06pq 69.53 ± 0.06h 13.13 ± 0.06pqr 15.43 ± 0.10lm
II-35 30.26 ± 0.15g 2.97 ± 0.06o 21.39 ± 0.10l 5.91 ± 0.06tu 0.00 ± 0.03pq 0.00 ± 0.00u 0.51 ± 0.06u 10.11 ± 0.03pqr
II-36 61.93 ± 0.10e 72.88 ± 0.06e 68.98 ± 0.15f 62.37 ± 0.06e 61.38 ± 0.03d 77.25 ± 0.06f 62.63 ± 0.06e 65.43 ± 0.03cd
Norcantharidin 88.17 ± 1.87a 34.31 ± 1.69g 86.64 ± 0.80b 17.72 ± 1.21j 20.00 ± 1.10ij 24.46 ± 1.13i 1.98 ± 2.24l 29.75 ± 1.27g
Cantharidin 88.16 ± 1.39a 38.54 ± 2.68f 100.00 ± 0.00a 10.19 ± 1.61k 67.59 ± 1.06cd 84.54 ± 1.39d 70.20 ± 0.84d 68.05 ± 1.29c
Thiabendazole 86.84 ± 1.22a 100.00 ± 0.00a 100.0 ± 0.00a 66.67 ± 0.00e 47.58 ± 1.60f 100.00 ± 0.00a 15.14 ± 1.18j 24.99 ± 1.17h
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* 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.

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 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.

Table 2.

Antifungal activity of some compounds against eight pathogens *.

Compd. IC50 (μg/mL) (CI 95%) **
V. m. B. b. S. f. G. c. A. a. S. s. A. s. C. s.
II-1 32.0800
(26.1510–39.3533)		 31.2568
(23.7127–41.2012)		 14.6778
(10.7872–19.9716)		 36.3526
(29.1302–45.3658)		 42.2537
(33.7577–52.8879)		 7.2482
(2.9912–17.5637)		 30.1335
(23.3387–38.9065)		 22.7788
(16.0894–32.2493)
II-4 27.8016
(22.1312–34.9249)		 24.8446
(18.2585–33.8063)		 10.6665
(7.6148–14.9412)		 16.7764
(11.7587–23.9352)		 31.4258
(24.6239–40.1066)		 4.2858
(1.6648–11.0334)		 14.0938
(9.1043–21.8178)		 10.4051
(12.9130–24.7109)
II-5 20.1058
(14.8666–27.1914)		 19.8375
(14.7821–26.6217)		 2.0791
(1.3098–3.3003)		 11.2505
(6.9283–18.2691)		 18.1002
(13.1268–24.9579)		 3.4556
(1.6958–7.0418)		 10.4753
(6.7490–16.2590)		 10.4051
(6.4109–16.8880)
II-8 11.3756
(7.2790–17.7778)		 13.6528
(9.8721–18.8814)		 0.8805
(0.4243–1.8275)		 7.7364
(3.8335–15.6128)		 13.8916
(9.3377–20.6664)		 0.9698
(0.4790–1.9633)		 5.1863
(2.5133–10.7020)		 7.5908
(4.4564–12.9298)
II-11 17.0896
(11.9965–23.3450)		 17.0640
(12.6223–23.0689)		 2.3717
(1.4367–3.9152)		 9.1071
(5.1797–16.0125)		 22.1465
(16.7792–29.2308)		 2.1783
(0.9142–5.1904)		 12.1081
(7.9591–18.4200)		 12.9298
(8.9552–18.6685)
II-14 47.7810
(39.0992–58.39.4)		 37.3895
(28.8394–48.4746)		 21.9666
(16.3757–29.4664)		 57.0963
(45.2452–72.0517)		 49.1546
(39.1730–61.6795)		 9.6192
(4.2886–21.5754)		 34.8290
(27.5093–44.0964)		 29.8409
(16.0894–32.2493)
Norcantharidin 17.1862
(9.2272–32.0104)		 141.8133
(61.1287–328.9946)		 17.1673
(10.0952–29.1937)		 465.1719
(86.5242–2500.8598)		 394.9566
(73.9654–2108.9689)		 54.5899
(32.0147–93.0839)		 594.2606
(211.1064–1672.8327)		 NA ***
Cantharidin 21.9320
(13.3453–36.0436)		 147.4225
(46.8714–463.6810)		 2.2175
(0.6116–12.0751)		 155.0286
(76.7546–313.1261)		 14.0428
(5.6729–34.7622)		 14.0622
(8.0013–24.7142)		 28.5223
(18.6015–43.7340)		 18.3900
(8.3854–40.3309)
TBZ 0.5191
(0.0218–12.3413)		 15.5378
(8.8501–27.2793)		 3.1998
(1.2133–8.4391)		 22.3806
(10.4428–47.9653)		 47.8696
21.2217–107.9790		 1.2947
(0.6661–2.5165)		 1268.5889
(110.6555–1453.4930)		 621.7874
60.1464–6427.9804
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* 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.

Figure 3.

Figure 3

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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

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 the phenyl ring plays an important role in activity, as compounds substituted at the C-4′ position (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.

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 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, ≥98%) was isolated from M. phalerata (Chinese blister beetle), bought from the Chinese herbal medicine market in Xian, China. Norcantharidin (NCTD, ≥98%) was purchased from Alfa Aesar Chemical Co. Ltd., (Haverhill, MA, USA). Dimethyl sulfoxide (DMSO, ≥99%) was obtained from J & K China Chemical Ltd. (Beijing, China). The fungicide thiabendazole (TBZ, ≥99.1%) 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: 164–165 °C; 1H-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, 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), 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: 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, 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: 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 (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′), 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, 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, 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), 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 (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′), 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, 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, 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 (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, 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, 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 (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, 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), 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), 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, 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, mp: 177–178 °C; 1H-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, 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 (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, 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), 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), 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 (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 (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, 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), 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, 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, mp: 169–171 °C; 1H-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, 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 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, 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, 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), 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, 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′), 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, 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), 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.

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 (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 (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, 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.

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, 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 (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, 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, 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), 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′), 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, 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]+) 300.0848, found 300.0851.

3-((4-Carboxyphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-17). Yield 44%, white solid, mp: 256–257 °C; 1H-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, 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, 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′) 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 solid, mp: 151–152 °C; 1H-NMR δ: 1.52–1.72 (m, 4H, H-1, 2), 3.05–3.17 (m, 2H, H-4, 5), 3.83 (s, 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′), 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′), 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 solid, mp: 165–166 °C; 1H-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, 1.89 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, 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), 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, found 314.0983.

3-((4-Methoxyphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-20). Yield 71%, white solid, mp: 167–168 °C; 1H-NMR: 1.46–1.69 (m, 4H, H-1, 2), 2.94 (d, J = 9.77 Hz, 1H, H-4), 3.03 (d, 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, 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); 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), 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, 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, 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 (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 solid, mp: 164–166 °C; 1H-NMR δ: 1.46–1.70 (m, 4H, H-1, 2), 3.00 (d, J = 9.46 Hz, 1H, H-4), 3.09 (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 (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.

3-((2-Nitrophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-23). Yield, 47%, yellow solid, mp: 155–156 °C; 1H-NMR δ: 1.46–1.72 (m, 4H, H-1, 2), 2.98–3.12 (m, 2H, H-4, 5), 4.63–4.85 (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 (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 (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 solid, mp: 178–179 °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), 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, found 329.0771.

3-((4-Nitrophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-25). Yield, 54%, yellow solid, mp: 156–157 °C; 1H-NMR δ: 1.48–1.70 (m, 4H, H-1, 2), 3.02 (d, J = 9.46 Hz, 1H, H-4), 3.12 (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), 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′), 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, 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.

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, 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, 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.

3-((4-Iodo-2-methylphenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-28). Yield, 56%, white solid, mp: 147–149 °C; 1H-NMR δ: 1.59 (br, s, 4H, H-1, 2), 2.26 (s, 3H, H-7′), 3.04–3.10 (m, 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′), 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, 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, 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 solid, mp: 156–157 °C; 1H-NMR δ: 1.47–1.68 (m, 4H, H-1, 2), 2.94 (d, J = 9.77 Hz, 1H, H-4), 3.05 (d, 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, 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, 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, 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.

3-((2-Methyl-3-nitrophenyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-31). Yield, 35%, white solid, mp: 144–145 °C; 1H-NMR δ: 1.59 (br, s, 4H, H-1, 2), 2.26 (s, 3H, H-7′), 3.04–3.10 (m, 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′), 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, 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, 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), 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 (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 (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.

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, 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, 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, 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, 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, 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.

3-((1H-Benzo[d]imidazol-2-yl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (II-36). Yield, 45%, white solid, mp: 189–190 °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, 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, 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), 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 (%) = [(dc − d0) − (dt − d0)]/(dc − d0) × 100 (1)

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. 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 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 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.

Footnotes

Sample Availability: Samples of the compounds II (1–36) are available from the authors.

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