Synthesis and Antioxidant Activity of New Norcantharidin Analogs

Article abstract of DOI:10.1002/cbdv.201800673

New norcantharidin analogs were designed and obtained as compounds with biological activity. As a starting material, exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid anhydride was used. Three groups of compounds: dicarboximides, triazoles and thiazolidines were obtained in multistep reactions. The ¹H- and ¹³C-NMR spectra were used to confirm the structures of all obtained products and they were in agreement with the proposed structure of substances. All derivatives were screened for their antioxidant activity. The most promising group was dicarboximides (1–4, 6). Derivatives 2–4 displayed antioxidant activity with EC₅₀=7.75–10.89 μg/ml, which may be comparable to strong antioxidant Trolox (EC₅₀=6.13 μg/ml). Excellent activity with EC₅₀=10.75 μg/ml also presented norcantharidin analog with 1,2,4-triazole system (12).

Full text of DOI:10.1002/cbdv.201800673

DOI: 10.1002/cbdv.201800673
FULL PAPER
Synthesis and Antioxidant Activity of New Norcantharidin Analogs
Anna Pachuta-Stec,*a Renata Nowak,b Wioleta Pietrzak,b and Monika Pituchaa
a
Independent Radiopharmacy Unit, Faculty of Pharmacy with Medical Analytics Division, Medical University of
Lublin, 4 A Chodźki Street, PL-20-093 Lublin, Poland, e-mail: anna.pachuta@umlub.pl
Department of Pharmaceutical Botany, Faculty of Pharmacy with Medical Analytics Division, Medical University
b
of Lublin, 1 Chodźki Street, PL-20-093 Lublin, Poland
New norcantharidin analogs were designed and obtained as compounds with biological activity. As a starting
material, exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid anhydride was used. Three groups of compounds:
1
dicarboximides, triazoles and thiazolidines were obtained in multistep reactions. The H- and 13C-NMR spectra
were used to confirm the structures of all obtained products and they were in agreement with the proposed
structure of substances. All derivatives were screened for their antioxidant activity. The most promising group
was dicarboximides (1–4, 6). Derivatives 2–4 displayed antioxidant activity with EC50 =7.75–10.89 μg/ml, which
may be comparable to strong antioxidant Trolox (EC50 =6.13 μg/ml). Excellent activity with EC50 =10.75 μg/ml
also presented norcantharidin analog with 1,2,4-triazole system (12).
Keywords: norcantharidin analogs, heterocycles, cyclization reaction, antioxidant activity, structure–activity
relationship, biological activity.
against OS by reacting with free radicals, chelating
catalytic metals and also by acting as oxygen
Introduction
Reactive oxygen species (ROS) are by-products of scavengers.[5,6] Searching for the compounds, which
enzymatic reactions occurring in the organism. They better protect and prevent oxidative stress, is a very
are produced during endogenous processes such as important issue in modern medicinal chemistry.
cell respiration, phagocytosis, biosynthesis, catalysis
Norcantharidin (exo-7-oxabicyclo[2.2.1]heptane-2,3-
and biotransformation. They can also be produced by dicarboxylic acid anhydride) is a terpenoid compound
exogenous processes (radiation, sunlight, heavy met- used as an anticancer drug in China. The first
als, bacteria, fungi, protozoa and viruses). Oxidative application of this compound was hepatoma, carcino-
processes taking place under the influence of the ROS mas of esophagus and gastric cardia.[7,8] In the recent
cause destruction of cells and tissue.[1] ROS are also years, special attention was paid to the mechanism of
very important factors in the aging processes, oxida- action of norcantharidin (NCTD), because it is compli-
tive stress (OS) and in the pathogenesis of various cated and still poorly elucidated. Chang et al. demon-
diseases. Among these diseases are cancer, rheuma- strated that NCTD induced cytotoxic activity against
toid arthritis, various neurodegenerative and pulmo- HepG2 by apoptosis, which is mediated through
nary diseases, atherosclerosis and DNA damage.[2,3] reactive oxygen species (ROS) generation and mito-
Probably, OS is the result of imbalance between chondrial pathway.[9] This anhydride also displayed
production of free radicals and the speed of their proliferative activity against these cells.[10] Similar
neutralization in the body.[3,4] Antioxidants are natural results were obtained for TSGH 8301 human urinary
or synthetic compounds, which play the shielding role bladder carcinoma cells.[11] NCTD promoted ROS
production and decreased levels of mitochondrial
membrane potential. In the case of DU145 human
prostate cancer cells, NCTD caused the reduction of
expression of proliferating cell nuclear antigen, disor-
Supporting information for this article is available on the
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Chem. Biodiversity 2019, 16, e1800673
der of mitochondrial membrane potential, deficit of Results and Discussion
ATP, induction of ROS and manganese superoxide
dismutase, activation of adenosine 5’-monophosphate,
Chemistry
which activated protein kinase and released cyto- In the present work, exo-7-oxabicyclo[2.2.1]heptane-
chrome c. All of these factors led to death of DU145 2,3-dicarboxylic acid anhydride (NCTD) was used as an
cells by apoptosis.[12] Other mechanism of action was initial material. This compound was heated with 4-
presented for NCTD by Yeh et al.[13] In this study, substitued 3-thiosemicarbazide in dry chloroform and
hepatocellular carcinoma (HCC) was used. NCTD new N-(substituted-thioureido)aminobicyclo dicarbox-
inhibited expression of proteases MMP-9 and u-PA imides were synthesized. Intramolecular cyclization
through the phosphorylation signaling pathway of reaction of dicarboximide with sodium hydroxide led
ERK1/2 and NF- κB. Additionally, NCTD revealed a to formation of 3,4-disubstituted 1,2,4-triazoline-5-
strong antimetastatic potential due to increased thione. In this reaction, the cyclic imide ring was
expression of degradation inhibitors ECM, which opened[20] and then spontaneous cyclization of the
reduced the ability of cell mobility of Huh7.[13] NCTD is linear chain of thiosemicarbazide took place, giving
also active in vitro and in vivo against multiple myelo- compounds with 1,2,4-triazole system.[15,21] Derivatives
ma through the NF-κB signal pathway.[14] Not only of 1,3-thiazolidine were prepared by Hantzsch reac-
NCTD but also derivatives of NCTD presented anti- tion, in which the sulfur atom of thioureido group
cancer activity. There are norcantharidin analogs with attacked the halogen atom of α-halocarbonyl com-
cyclic (lactone and imides ring) and with opening ring pound and then elimination of hydrogen bromide and
of the anhydride (amide, amide-acid and mono- water led to the cyclic thiazole ring.[22] The thioureido
esterified analogs). All these derivatives have anti- group of dicarboximide, which has two nucleophilic
cancer activity against liver carcinoma (HepG2 and centers, reacted with ethoxycarbonylmethyl group of
Hep3B), colon cancer (HT29) and glioblastoma (SJ- ethyl bromoacetate, and then, intermediate for further
G2).[15–17] Some of them are strong inhibitors of cyclization was produced. The reaction proceeded in
protein phosphatases PP1 and PP2.[18,19] Recently, one step without isolation of intermediate.[23] Two
Peksel et al. examined some derivatives of NCTD and different structural isomers I and II can be obtained in
bridged perhydroisoindole for their antioxidant and this synthesis (Scheme 1). Based on the previous article,
radical scavenging activities.[5] Most of the derivatives it can be speculated that only one structural isomer I
presented significant antioxidant and radical scaveng- was obtained.[24] In conclusion, the structures of the
ing activities. On the basis of the above mentioned products 14–20 were determined based on previous
studies, we can state that in the future NCTD and discussion of the structures of similar compounds[24]
derivatives of NCTD may be used in the prevention and results are consistent with the description in
and treatment of cancer and this can be important in literature.[23,25–27] The synthetic pathway is presented
the prevention of secondary cancers.[9]
in Scheme 2.
Unfortunately, in the case of the reaction of 4-(4-
In the light of the above research and according
with our sustained interest in the application of chlorophenyl)-3-thiosemicarbazide with NCTD, pure
different dicarboxylic acid anhydride, especially NCTD dicarboximide was not isolated, but further cyclization
in the synthesis of biologically active compounds, we of this derivative led to 1,2,4-triazole (9) and 1,3-
designed and obtained a new class of norcantharidin thiazolidine (16) with good purity. 1-(1,3-Dioxooctahy-
analogs such as dicarboximides, triazoles and thiazoli- dro-2H-4,7-epoxyisoindol-2-yl)-3-phenylthiourea
(1)
dines. Study of the antioxidant activities of NCTD and was previously obtained by other authors.[28] The
its derivatives is very important, so we examined spectral characterization of compound 1, which we
prepared derivatives for these activity.
have synthesized, is consistent with the published
The newly obtained compounds can be applied in data.
practice. The modification of the structure of the
1
The H- and 13C-NMR spectra were used to confirm
parent derivatives may provide a promising direction the structures of all the obtained products and they
to obtain new biologically active norcantharidin ana- were in agreement with the proposed structure of
1
logs. These studies can also help medicinal chemists, substances 1–20. The H-NMR spectra of compounds
biochemists and pharmacologists anticipate superior 1–6 presented two singlets for two NH groups
activity and synthesize unknown compounds with between 8.13–9.91 ppm and 9.80–10.37 ppm. In the
improved biological effect compared to the derivatives 13C-NMR spectra of these compounds (1–6), typical
obtained in this work.
signals corresponding to cyclic carboximides C=O
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Chem. Biodiversity 2019, 16, e1800673
Scheme 1. Probable mechanism leading to the formation of 1,3-thiazolidine.
Scheme 2. Synthesis of compounds 1–20.
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Chem. Biodiversity 2019, 16, e1800673
groups and C=S group of linear chain were observed.
Table 1. Antiradical activity of synthesized compounds 1–20.
They resonated between 174.61–175.20 ppm and
180.98–181.78 ppm, respectively. The 1,2,4-triazole-5-
thiones (7–12) in the 1H-NMR spectra presented
characteristic singlet signal for the NH group at 11.97–
12.35 ppm. In the 13C-NMR spectra for these deriva-
tives, the signal corresponding to the C=S group
appeared at 171.71–171.96 ppm. In the 1H-NMR
spectra for 1,3-thiazolidine (14–20), characteristic
signals due to the CH2 protons appeared as singlet at
4.14–4.36 ppm, whereas 13C-NMR presented signals
due to the C=O group of thiazolidine ring at 171.29–
172.40 ppm.
Compound Antioxidant activity
*
*
DPPH
ABTS+
TEAC
EC50 (range
EC50
from 0 to 5)
0 – inactive,
5 – very active
[mM TroloxgÀ 1
compound][a]
[μgmLÀ 1 [a]
]
1
2
3
4
5
6
7
8
5
4
5
4
2
3
4
2
4
3
nd
3
nd
0/1
0/1
0/1
0/1
0/1
nd
0/1
0.989�0.025
2.750�0.033
2.256�0.095
3.173�0.140
0.163�0.009
0.969�0.029
1.303�0.049
1.444�0.046
1.582�0.017
1.665�0.036
nd
24.83�0.62
8.93�0.11
10.89�0.46
7.75�0.35
150.49�7.81
25.35�0.77
18.86�0.71
17.02�0.53
15.52�0.17
14.75�0.32
nd
9
Antiradical Activity
10
11
12
13
14
15
16
17
18
19
20
All the synthesized compounds were primary screened
for their antioxidant activity using DPPH-TLC assay.
The most active derivatives 1–10, 12, 14–18 and 20
were selected for antiradical activity analysis using an
2.284�0.056
10.75�0.27
–
–
–
–
–
nd
–
–
-
–
–
–
nd
–
*
improved ABTS+ decolorization assay. Among all
examined substances, the most promising group were
dicarboximides (1–4, 6), whereas compounds with
1,3-thiazolidine moiety (14–18, 20) were inactive.
Excellent antioxidant activity was displayed by dicar-
boximides 2–4 with EC50 =7.75–10.89 μg/ml, which
may be comparable to strong antioxidant Trolox
(EC50 =6.13 μg/ml). The dicarboximide derivatives 1
and 6 possess quite good activity with EC50 =24.83–
25.35 μg/ml. Only the dicarboximide 5 bearing ethyl
[a]
Values are expressed as means of three replicates�SD. ‘–’,
no activity; ‘nd’, not determined; EC50 for Trolox=
6.13�0.28 μgmLÀ 1
.
substituent was inactive. Interestingly, derivative 12 Structure-Activity Relationship
with ethyl substituent at 4-position of triazole ring,
which was obtained from the inactive compound 5, From the antiradical activity analysis, it can be stated
also presented excellent activity with EC50 =10.75 μg/ that the initial compound, exo-7-oxabicyclo[2.2.1]-
ml. The other 1,2,4-triazoles 7–10 indicated good heptane-2,3-dicarboxylic acid anhydride (NCTD), had
antioxidant activity (EC50 =15.52–25.35 μg/ml). With no antioxidant activity, whereas the dicarboximide
regards to chelating power (CHEL), the highest activity derivative obtained from NCTD and various 4-substi-
showed compound 6, but only 36% inhibition was tuted 3-thiosemicarbazides indicated good to excel-
determined. The remaining compounds were inactive. lent activity. Among them, the most active were
The antiradical activity of synthesized compounds (1– compounds 2–4 with aryl substituent (2-methylphen-
20) is presented in Table 1.
yl, 2-chlorophenyl and 4-fluorophenyl) attached to
Some derivatives of NCTD and bridged perhydroi- nitrogen of thioureido moiety. Compound 1 with
soindole were also evaluated for their antioxidant and unsubstituted phenyl group attached to nitrogen of
radical scavenging activities by Peksel et al.[5] Examined thioureido moiety displayed only good activity. In the
compounds presented lower radical scavenging activ- case of dicarboximide derivatives with aromatic sub-
ities (IC50 =25.6–130.6 μg/ml) when compared to stituents, antioxidant activity can be connected with
commercial antioxidants, for example Trolox (IC50 = the presence of substituents attached to phenyl
4.5 μg/ml).
moiety at 2- or 4-position. It can be stated that the
character of substituent (electron donating or with-
drawing) does not affect activity. Dicarboximide de-
rivatives with aliphatic substituents: ethyl (in 5) or
morpholinylethyl (in 6) at nitrogen of thioureido
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Chem. Biodiversity 2019, 16, e1800673
Figure 1. Structure of compounds additionally examined for antiradical activity.
moiety had no activity or presented good activity,
Conclusions
respectively. Closer inspection of these compounds
revealed that better activity showed derivative 6 with In this work, 20 analogs of norcantharidin were
a bulky aliphatic substituent. Interestingly, when obtained. Among them, there were dicarboximides
corresponding inactive dicarboximide bearing ethyl (1–6), triazoles (7–13) and thiazolidines (14–20). All
substituent was cyclized to 3,4-disubstituted 1,2,4- the synthesized compounds were evaluated for their
triazoline-5-thione, the obtained compound 12 re- antioxidant activity. Generally, new triazoles (7–13)
vealed excellent antiradical activity. In this case, the improved their antiradical activity compared to the
cyclization reaction improved activity. Derivative 7 derivatives obtained previously.[5] The introduction of
with unsubstituted phenyl at 4-position of 1,2,4-triazo- the carboxylic group into the triazole ring increased
line-5-thione system in comparison to dicarboximide 1 the activity. The most promising group were dicarbox-
with the same substituent showed improved activity, imides, especially compounds 2–4 with EC50 =7.75–
too. Three other 1,2,4-triazoline-5-thione derivatives 10.89 μg/ml. Similar activity with EC50 =10.75 μg/ml
8–10 did not improve their activity in comparison to displayed 3-(4-ethyl-4,5-dihydro-5-thioxo-1H-1,2,4-tria-
counterpart dicarboximides 3–4.
zol-3-yl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
For a more accurate SAR analysis, some compounds (12). The antioxidant activity of these compounds was
with a similar structure, which were obtained previ- comparable to commonly used antioxidant Trolox.
ously as anticancer agents, were also tested.[29] Derivatives with the best antiradical activity may be a
Structures of these derivatives is presented in Figure 1. promising lead for future investigation. Additionally,
Among them, there were NCTD, ethyl N-(1,3-dioxooc- the most active compounds, dicarboximides (2–4) and
tahydro-2H-4,7-epoxyisoindol-2-yl)carbamimidothioate triazole 12, may be promising candidates for therapeu-
(A) and N-substituted amides of 3-[3-(ethylsulfanyl)- tic applications.
1H-1,2,4-triazol-5-yl]-7-oxabicyclo[2.2.1]heptane-2-car-
boxylic acid (B–D).
In the case of 1,2,4-triazoles (B–D) with the amide Experimental Section
and ethylsulfanyl group from Figure 1, no antiradical
activity was observed, whereas 1,2,4-triazoline-5-thi-
Chemistry
one 7–10 and 12 with carboxylic and thione group All the used chemicals were purchased from Sigma-
showed good to excellent activity. This fact suggested Aldrich (Munich, Germany) or Merck Co. (Darmstadt,
that the carboxylic group and thione form of triazole Germany) and applied without further purification.
were beneficial in terms of bioactivity. In general, the Melting points were determined in a Fischer-Johns
1
introduction of unsubstituted phenyl, methylphenyl, block and presented without any corrections. H- and
chlorophenyl or fluorophenyl substituent to dicarbox- 13C-NMR spectra were recorded on a Bruker Avance
imide and 1,2,4-triazoline-5-thione is also beneficial in 300 MHz spectrometer in (D6)DMSO; δ in ppm relative
terms of antiradical activity. Unfortunately, in the case to Me4Si as internal standard, J in Hz. MS spectra were
of 1,3-thiazolidine derivatives (14–18, 20), probably, recorded on Bruker microTOF-Q II and processed using
the presence of thiazolidine moiety (five-membered Compass Data Analysis software; in m/z. Elemental
heterocyclic ring with two heteroatoms of sulfur and analysis was made on a PerkinElmer 2400 CHN
nitrogen) can be connected with lack of antiradical analyzer. All results were in good agreement with
activity irrespectively of substituent. All these results calculated values. The error range of �0.4% was for
provide useful information about structure–activity each element analyzed.
relationships.
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Chem. Biodiversity 2019, 16, e1800673
General Method for the Synthesis of N-(Substituted-
1-(1,3-Dioxooctahydro-2H-4,7-epoxyisoindol-2-
°
thioureido)aminobicyclo Dicarboximide (1–6). 10 mmol yl)-3-ethylthiourea (5). Yield: 93%. M.p. 170–172 C.
of exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid 1H-NMR (300 MHz, (D6)DMSO): 1.03 (t, J=6.9, Me);
anhydride and 10 mmol of appropriate 4-substituted 1.65–1.72 (m, 2CH2); 3.11 (s, 2CH); 3.42 (q, J=6.9,
3-thiosemicarbazide in dry chloroform were refluxed CH2Me); 4.71 (s, CHÀ OÀ CH); 8.22 (br. s, NH); 9.80 (s,
for 1 h. After cooling, the precipitate was filtered off NH). 13C-NMR (75 MHz, (D6)DMSO): 14.27 (Me); 28.40
and crystallized from ethanol or ethanol-water.
(2CH2); 38.20 (CH2Me); 48.51 (2CH); 79.08 (CHÀ OÀ CH);
175.20 (2C=O); 180.98 (C=S). HR-MS: 270.0691 ([M+
H]+, C11H15N3O3S+; calc. 269.3201).
1-(1,3-Dioxooctahydro-2H-4,7-epoxyisoindol-2-
°
yl)-3-phenylthiourea (1). Yield: 80%. M.p. 190–192 C.
1H-NMR (300 MHz, (D6)DMSO): 1.66–1.73 (m, 2CH2);
1-(1,3-Dioxooctahydro-2H-4,7-epoxyisoindol-2-
3.17 (s, 2CH); 4.75 (s, CHÀ OÀ CH); 7.17–7.36 (m, Ph); yl)-3-[2-(morpholin-4-yl)ethyl]thiourea (6). Yield:
9.91 (br. s, NH); 10.22 (s, NH). 13C-NMR (75 MHz, (D6) 87%. M.p. 180–182 C. H-NMR (300 MHz, (D6)DMSO):
1
°
DMSO): 28.52 (2CH2); 48.68 (2CH); 78.93 (CHÀ OÀ CH); 1.59 (s, 2CH2); 2.38 (s, CH2À NÀ CH2 +CH2); 3.12 (s, 2CH);
125.90 (2 o-PhCH); 126.13 (p-PhCH); 128.91 (2 m- 3.58 (s, CH2À OÀ CH2 +CH2); 4.72 (s, CHÀ OÀ CH); 8.13 (br.
PhCH); 138.99 (i-PhC); 174.74 (2C=O); 181.21 (C=S). HR- s, NH); 9.92 (s, NH). 13C-NMR (75 MHz, (D6)DMSO): 28.48
MS: 318.0597 ([M+H]+, C15H15N3O3S+; calc. 317.3629). (2CH2); 41.78 (CH2); 48.57 (2CH); 53.77 (CH2À NÀ CH2);
56.86 (CH2); 66.60 (CH2À OÀ CH2); 78.98 (CHÀ OÀ CH);
1-(2-Chlorophenyl)-3-(1,3-dioxooctahydro-2H-
174.84 (2C=O); 181.50 (C=S). HR-MS: 353.0206 ([M+
4,7-epoxyisoindol-2-yl)thiourea (2). Yield: 86%. M.p. H]+, C15H22N4O4S+; calc. 354.4246).
186–188 C. 1H-NMR (300 MHz, (D6)DMSO): 1.70 (s,
°
2CH2); 3.15 (s, 2CH); 4.75 (s, CHÀ OÀ CH); 7.18–7.64 (m,
General Method for the Synthesis of 3,4-Disubstituted
Ph); 9.83 (br. s, NH); 10.37 (s, NH). 13C-NMR (75 MHz, 1,2,4-Triazoline-5-thione (7–13). A solution of N-(sub-
(D6)DMSO): 28.52 (2CH2); 48.90 (2CH); 78.97 stituted-thioureido)aminobicyclo
dicarboximide
(CHÀ OÀ CH); 127.87 (o-PhCH); 129.20 (p-PhCH); 130.00 (2 mmol) in 2% sodium hydroxide (5 ml) was refluxed
(m-PhCH); 131.85 (m-PhCH); 132.22 (o-PhCÀ Cl); 136.39 for 2 h. After cooling, the mixture was filtered off and
(i-PhC); 174.61 (2C=O); 181.78 (C=S). HR-MS: 352.0218 the isolated solution was acidified with 3 M HCl. The
([M+H]+, C15H14ClN3O3S+; calc. 351.8080).
precipitate was filtered off and crystallized from
ethanol.
1-(1,3-Dioxooctahydro-2H-4,7-epoxyisoindol-2-
yl)-3-(4-fluorophenyl)thiourea (3). Yield: 95%. M.p.
3-(4,5-Dihydro-4-phenyl-5-thioxo-1H-1,2,4-tria-
1
°
174–176 C. H-NMR (300 MHz, (D6)DMSO): 1.53–1.83 zol-3-yl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic
(m, 2CH2); 3.17 (s, 2CH); 4.79 (s, CHÀ OÀ CH); 7.03–7.68 Acid (7). Yield: 70%. M.p. 260–262 C. 1H-NMR
°
(m, Ph); 9.90 (br. s, NH); 10.27 (s, NH). 13C-NMR (300 MHz, (D6)DMSO): 1.37–1.56 (m, 2CH2); 3.41–3.45
(75 MHz, (D6)DMSO): 28.52 (2CH2); 48.71 (2CH); 78.95 (m, 2CH); 4.77 (s, CHÀ OÀ CH); 7.39–7.60 (m, Ph); 12.29
(CHÀ OÀ CH); 115.49 (2 m-PhCH); 126.74 (o-PhCH); (br. s, NH); 13.63 (s, COOH). 13C-NMR (75 MHz, (D6)
129.07 (o-PhCH); 135.26 (i-PhC); 158.68 (p-PhCÀ F); DMSO): 28.21 (CH2); 29.63 (CH2); 43.50 (CH-triazole);
161.89 (p-PhC–F); 174.75 (2C=O); 181.54 (C=S). HR-MS: 53.87
(CHÀ COOH);
78.02
(CHÀ OÀ CH);
79.72
336.0531 ([M+H]+, C15H14FN3O3S+; calc. 335.3534).
(CHÀ OÀ CH); 129.14 (2 o-PhCH); 129.77 (2 m-PhCH+p-
PhCH); 134.27 (i-PhC); 152.78 (Ctriazole); 167.31 (COOH);
1-(1,3-Dioxooctahydro-2H-4,7-epoxyisoindol-2-
171.77
(C=S).
HR-MS:
318.0613
([M+H]+,
yl)-3-(2-methylphenyl)thiourea (4). Yield: 83%. M.p. C15H15N3O3S+; calc. 317.3629).
1
°
200–202 C. H-NMR (300 MHz, (D6)DMSO): 1.64–1.76
(m, 2CH2); 2.13 (br. s, Me), 3.15 (s, 2CH); 4.75 (s,
3-[4-(2-Chlorophenyl)-4,5-dihydro-5-thioxo-1H-
CHÀ OÀ CH); 7.06–7.28 (m, Ph); 9.68 (br. s, NH); 10.21 (s, 1,2,4-triazol-3-yl]-7-oxabicyclo[2.2.1]heptane-2-car-
NH). 13C-NMR (75 MHz, (D6)DMSO): 17.87 (Me); 28.53 boxylic Acid (8). Yield: 65%. M.p. 260–262 C. H-NMR
1
°
(2CH2); 48.89 (2CH); 78.93 (CHÀ OÀ CH); 126.64 (o- (300 MHz, (D6)DMSO): 1.29–1.66 (m, 2CH2); 3.45–3.47
PhCH); 127.73 (p-PhCH); 129.29 (m-PhCH); 130.79 (m- (m, 2CH); 4.80 (s, CHÀ OÀ CH); 7.29–7.86 (m, Ph); 12.30
PhCH); 136.30 (o-PhCÀ Me); 137.51 (i-PhC); 174.91 (br. s, NH); 13.76 (s, COOH). 13C-NMR (75 MHz, (D6)
(2C=O); 181.15 (C=S). HR-MS: 332.0755 ([M+H]+, DMSO): 28.16 (CH2); 29.59 (CH2); 43.53 (CH-triazole);
C16H17N3O3S+; calc. 331.3895).
54.12
(CHÀ COOH);
77.70
(CHÀ OÀ CH);
79.88
(CHÀ OÀ CH); 128.92 (o-PhCH); 130.97 (p-PhCH); 131.73
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(o-PhCÀ Cl); 132.09 (m-PhCH); 132.28 (m-PhCH); 132.81 (75 MHz, (D6)DMSO): 13.90 (Me); 28.34 (CH2); 29.65
(i-PhC); 152.82 (Ctriazole); 167.32 (COOH); 171.71 (C=S). (CH2); 38.51 (CH2); 43.15 (CH-triazole); 53.89
HR-MS: 352.0228 ([M+H]+, C15H14ClN3O3S+; calc. (CHÀ COOH); 78.00 (CHÀ OÀ CH); 79.52 (CHÀ OÀ CH);
351.8080).
152.44 (Ctriazole); 165.74 (COOH); 171.81 (C=S). HR-MS:
270.0658 ([M+H]+, C11H15N3O3S+; calc. 269.3201).
3-[4-(4-Chlorophenyl)-4,5-dihydro-5-thioxo-1H-
1,2,4-triazol-3-yl]-7-oxabicyclo[2.2.1]heptane-2-car-
3-[4,5-Dihydro-4-[2-(4-morpholinyl)ethyl]-5-
1
°
boxylic Acid (9). Yield: 62%. M.p. 160–162 C. H-NMR thioxo-1H-1,2,4-triazol-3-yl]-7-oxabicyclo[2.2.1]-
(300 MHz, (D6)DMSO): 1.30–1.67 (m, 2CH2); 2.77–3.12 heptane-2-carboxylic Acid (13). Yield: 66%. M.p.
(m, 2CH); 4.78 (s, CHÀ OÀ CH); 7.44–7.65 (m, Ph); 12.35 256–258 C. 1H-NMR (300 MHz, (D6)DMSO): 1.53 (s,
°
(br. s, NH); 13.68 (s, COOH). 13C-NMR (75 MHz, (D6) 2CH2); 2.95–3.35 (m, CH2); 3.36–3.52 (m, CH2À NÀ CH2);
DMSO): 28.14 (CH2); 29.62 (CH2); 43.42 (CH-triazole); 3.68–3.71 (m, CH2); 3.77–4.17 (m, CH2À OÀ CH2); 4.39–
53.96
(CHÀ COOH);
77.67
(CHÀ OÀ CH);
80.37 4.57 (m, 2CH); 4.90 (s, CHÀ OÀ CH); 11.97 (br. s, NH);
(CHÀ OÀ CH); 129.88 (2 o-PhCH); 131.10 (2 m-PhCH); 13.58 (s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 28.51
133.16 (p-PhCÀ Cl); 134.48 (i-PhC); 152.83 (Ctriazole); (CH2); 29.30 (CH2); 38.02 (CH2); 43.13 (CH-triazole);
167.28 (COOH); 171.86 (C=S). HR-MS: 352.0228 ([M+ 50.68 (CH2); 52.52 (CH2À NÀ CH2); 54.27 (CH–COOH);
H]+, C15H14ClN3O3S+; calc. 351.8080).
63.74 (CH2À OÀ CH2); 78.05 (CHÀ OÀ CH); 79.55
(CHÀ OÀ CH); 153.07 (Ctriazole); 165.05 (COOH); 171.96
(C=S). HR-MS: 355.1156 ([M+H]+, C15H22N4O4S+; calc.
3-[4-(4-Fluorophenyl)-4,5-dihydro-5-thioxo-1H-
1,2,4-triazol-3-yl]-7-oxabicyclo[2.2.1]heptane-2-car- 354.4246).
1
°
boxylic Acid (10). Yield: 72%. M.p. 245–247 C. H-
NMR (300 MHz, (D6)DMSO): 1.33–1.66 (m, 2CH2); 2.80–
General Method for the Synthesis of N-[(4-Oxo-3-
3.06 (m, 2CH); 4.78 (s, CHÀ OÀ CH); 7.32–7.58 (m, Ph); substituted-1,3-thiazolidin-2-yl)imino]-7-oxabicyclo-
12.25 (br. s, NH); 13.66 (s, COOH). 13C-NMR (75 MHz, [2.2.1]heptane Dicarboximide (14–20). To a suspension
(D6)DMSO): 28.16 (CH2); 29.62 (CH2); 43.43 (CH-triazole); of N-(substituted-thioureido)aminobicyclo dicarboxi-
53.90
(CHÀ COOH);
78.05
(CHÀ OÀ CH);
79.72 mide (2.5 mmol) in absolute ethanol (25 ml), anhy-
(CHÀ OÀ CH); 116.58 (2m-PhCH); 116.89 (2 m-PhCH); drous sodium acetate (10 mmol) and ethyl bromoace-
130.50 (i-PhC); 131.44 (o-PhCH); 131.57 (o-PhCH); tate were added and the mixture was refluxed for 4 h.
152.94 (Ctriazole); 160,97 (p-PhCÀ F); 164,41 (p-PhCÀ F); After cooling, the solution was allowed to stand
167.40 (COOH); 171.86 (C=S). HR-MS: 336.0524 ([M+ overnight. The precipitate was filtered off and crystal-
H]+, C15H14FN3O3S+; calc. 335.3534).
lized from ethanol.
3-[4,5-Dihydro-4-(2-methylphenyl)-5-thioxo-1H-
2-[(4-Oxo-3-phenyl-1,3-thiazolidin-2-ylidene)-
1,2,4-triazol-3-yl]-7-oxabicyclo[2.2.1]heptane-2-car- amino]hexahydro-1H-4,7-epoxyisoindole-1,3(2H)-
1
boxylic Acid (11). Yield: 68%. M.p. 168–170 C. H- dione (14). Yield: 86%. M.p. 282–284 C. 1H-NMR
NMR (300 MHz, (D6)DMSO): 1.31–1.69 (m, 2CH2); 2.12 (300 MHz, (D6)DMSO): 1.71 (s, 2CH2); 3.18 (s, 2CH); 4.33
(s, Me); 3.40–3.47 (m, 2CH); 4.73 (s, CHÀ OÀ CH); 7.18– (s, CH2); 4.76 (s, CHÀ OÀ CH); 7.30–7.75 (m, Ph). 13C-NMR
7.49 (m, Ph); 12.31 (br. s, NH); 13.69 (s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 28.43 (2CH2); 33.61 (CH2thiazol);
(75 MHz, (D6)DMSO): 17.82 (Me); 28.09 (CH2); 29.66 48.79 (2CH); 78.81 (CHÀ OÀ CH); 128.47 (2 o-PhCH);
(CH2); 43.41 (CH-triazole); 54.29 (CHÀ COOH); 77.66 129.38 (p-PhCH); 129.62 (2 m-PhCH); 134.70 (i-PhC);
(CHÀ OÀ CH); 79.84 (CHÀ OÀ CH); 127.65 (o-PhCH); 129.38 170.81 (Cthiazol); 171.98 (C=O); 172.73 (2C=O). HR-MS:
(p-PhCH); 130.44 (m-PhCH); 131.54 (m-PhCH); 133.12 358.0558 ([M+H]+, C17H15N3O4S+; calc. 357.3837).
(o-PhC–Me); 136.60 (i-PhC); 152.83 (Ctriazole); 166.71
°
°
(COOH); 171.73 (C=S). HR-MS: 332.0791 ([M+H]+,
2-{[3-(2-Chlorophenyl)-4-oxo-1,3-thiazolidin-2-
ylidene]amino}hexahydro-1H-4,7-epoxyisoindole-
1,3(2H)-dione (15). Yield: 62%. M.p. 238–240 C. H-
NMR (300 MHz, (D6)DMSO): 1.65 (s, 2CH2); 3.09 (s, 2CH);
C16H17N3O3S+; calc. 331.3895).
1
°
3-(4-Ethyl-4,5-dihydro-5-thioxo-1H-1,2,4-triazol-
3-yl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic Acid 4.36 (s, CH2); 4.69 (s, CHÀ OÀ CH); 7.47–7.75 (m, Ph). 13C-
1
°
(12). Yield: 60%. M.p. 260–262 C. H-NMR (300 MHz, NMR (75 MHz, (D6)DMSO): 28.43 (2CH2); 33.59
(D6)DMSO): 1.21 (t, J=6.0, Me); 1.50–1.84 (m, 2CH2); (CH2thiazol); 48.79 (2CH); 78.82 (CHÀ OÀ CH); 128.85 (o-
3.29–3.50 (m, 2CH); 3.88 (q, J=6.0, CH2Me); 4.80 (s, PhCH); 130.52 (p-PhCH); 131.34 (m-PhCH); 131.86 (m-
CHÀ OÀ CH); 12.35 (br. s, NH); 13.39 (s, COOH). 13C-NMR PhCH); 132.13 (o-PhCÀ Cl); 132.32 (i-PhC); 169.18
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Chem. Biodiversity 2019, 16, e1800673
(Cthiazol); 171.29 (C=O); 172.45 (2C=O). HR-MS: 392.0139
2-({3-[2-(Morpholin-4-yl)ethyl]-4-oxo-1,3-thiazo-
lidin-2-ylidene}amino)hexahydro-1H-4,7-epoxyiso-
([M+H]+, C17H14ClN3O4S+; calc. 391.8288).
indole-1,3(2H)-dione (20). Yield: 83%. M.p. 180–
1
°
2-{[3-(4-Chlorophenyl)-4-oxo-1,3-thiazolidin-2-
182 C. H-NMR (300 MHz, (D6)DMSO): 1.55 (s, 2CH2);
ylidene]amino}hexahydro-1H-4,7-epoxyisoindole-
2.36 (s, CH2À NÀ CH2); 2.58 (s, CH2); 3.13 (s, 2CH); 3.45 (s,
1
°
1,3(2H)-dione (16). Yield: 65%. M.p. 278–280 C. H- CH2À OÀ CH2); 3.83 (s, CH2); 4.14 (s, CH2); 4.62 (s,
NMR (300 MHz, (D6)DMSO): 1.62 (s, 2CH2); 3.12 (s, 2CH); CHÀ OÀ CH). 13C-NMR (75 MHz, (D6)DMSO): 28.46 (CH2);
4.22 (s, CH2); 4.70 (s, CHÀ OÀ CH); 7.33–7.52 (d, J=9.0, 33.19 (CH2thiazol); 40.05 (CH2); 48.84 (2CH); 53.60
Ph); 7.53–7.67 (d, J=9.0, Ph). 13C-NMR (75 MHz, (D6) (CH2À NÀ CH2); 54.11 (CH2); 66.64 (CH2À OÀ CH2); 78.77
DMSO): 28.43 (2CH2); 33.72 (CH2thiazol); 48.80 (2CH); (CHÀ OÀ CH); 169.95 (Cthiazol); 172.40(C=O); 172.83
78.82 (CHÀ OÀ CH); 129.69 (2 o-PhCH); 130.38 (2 m- (2C=O). HR-MS: 395.1036 ([M+H]+, C17H22N4O5S+;
PhCH); 133.49 (p-PhCÀ Cl); 133.96 (i-PhC); 170.62 calc. 394.4454).
(Cthiazol); 171.80 (C=O); 172.69 (2C=O). HR-MS: 392.0125
([M+H]+, C17H14ClN3O4S+; calc. 391.8288).
Antiradical Activity Analysis
2-{[3-(4-Fluorophenyl)-4-oxo-1,3-thiazolidin-2-
Preliminary assessment of the antioxidant activity of
synthesized compounds was screened using DPPH-
ylidene]amino}hexahydro-1H-4,7-epoxyisoindole-
1
1,3(2H)-dione (17). Yield: 69%. M.p. 282–284 C. H- TLC assay.[30,31] The solutions of analyzed compounds
NMR (300 MHz, (D6)DMSO): 1.65 (s, 2CH2); 3.11 (s, 2CH); at concentration 0.033 M were developed on a silica
4.22 (s, CH2); 4.80 (s, CHÀ OÀ CH); 7.27 – 7.56 (m, Ph). gel TLC plate (Merck, Darmstadt, Germany) using:
13C-NMR (75 MHz, (D6)DMSO): 28.43 (2CH2); 33.64 toluene/methanol/25% ammonia (9:1:0.05) solvent
(CH2thiazol); 48.80 (2CH); 78.82 (CHÀ OÀ CH); 116.43 (m- system. After drying, TLC plates were sprayed with
PhCH); 116.73 (m-PhCH); 130.81 (o-PhCH+i-PhC); 0.2% 1,1-diphenyl-2,2-picrylhydrazyl (DPPH, Fluka Co.,
130.88 (o-PhCH); 160,49 (p-PhCÀ F); 163,97 (p-PhCÀ F); Buchs, Switzerland) solution in methanol. Compounds
170.78 (Cthiazol); 171.94 (C=O); 172.71 (2C=O). HR-MS: showing a yellow-on-purple spot were regarded as
°
376.0440 ([M+H]+, C17H14FN3O4S+; calc. 375.3742).
antioxidants.
2-{[3-(2-Methylphenyl)-4-oxo-1,3-thiazolidin-2-
The antiradical activity was assayed using an
*
ylidene]amino}hexahydro-1H-4,7-epoxyisoindole-
improved ABTS+ decolorization assay with some
1
1,3(2H)-dione (18). Yield: 72%. M.p. 292–294 C. H- modifications.[32] ABTS was dissolved in water to a
°
*
NMR (300 MHz, (D6)DMSO): 1.64 (s, 2CH2); 2.21 (s, Me); 8.7 mM concentration. ABTS radical cation (ABTS+
)
3.09 (s, 2CH); 4.30 (s, CH2); 4.68 (s, CHÀ OÀ CH); 7.26 – was produced by reacting 1.25 ml of ABTS stock
7.41 (m, Ph). 13C-NMR (75 MHz, (D6)DMSO): 17.43 (Me); solution with 5 ml of potassium persulfate (12.2 mM)
28.43 (2CH2); 33.62 (CH2thiazol); 48.80 (2CH); 78.79 and allowing the mixture to stand in the dark at room
*
(CHÀ OÀ CH); 127.44 (o-PhCH); 129.02 (p-PhCH); 130.01 temperature for 12–18 h before use. The ABTS+
(m-PhCH); 131.29 (m-PhCH); 133.96 (o-PhCÀ Me); 136.44 solution was diluted with methanol (1:100).
(i-PhC); 169.85 (Cthiazol); 171.88 (C=O); 172.74 (2C=O).
HR-MS: 372.0678 ([M+H]+, C18H17N3O4S+; calc.
To determine EC50 of samples, the technique with
371.4103).
96-well microplates was used. Aliquots of 180 μl of a
*
ABTS+ solution were mixed with 20 μl of the samples
2-[(3-Ethyl-4-oxo-1,3-thiazolidin-2-ylidene)-
diluted to various concentrations in 96-well micro-
plates. Absorbance was recorded after 6 min of
amino]hexahydro-1H-4,7-epoxyisoindole-1,3(2H)-
dione (19). Yield: 76%. M.p. 182–184 C. 1H-NMR incubation at 734 nm using the Infinite Pro 200F
(300 MHz, (D6)DMSO): 1.22 (t, J=9.0, Me), 1.73 (s, microplate reader (Tecan Group Ltd., Männedorf,
°
2CH2); 3.41 (s, 2CH); 3.81 (q, J=9.0, CH2Me); 4.18 (s, Switzerland). The ability of the extract to quench the
*
CH2); 4.80 (s, CHÀ OÀ CH). 13C-NMR (75 MHz, (D6)DMSO): ABTS free radicals was determined using the following
12.28 (Me); 28.45 (2CH2); 33.39 (CH2thiazol); 38.32 (CH2); equation:
48.81 (2CH); 78.82 (CHÀ OÀ CH); 169.67 (Cthiazol); 172.11
Scavenging % ¼ ½ðAcÀ AaÞ=Ac� � 100
(C=O); 172.86 (2C=O). HR-MS: 310.0609 ([M+H]+,
C13H15N3O4S+; calc. 309.3409).
where Ac is the absorbance of control and Aa is the
absorbance of sample.
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Chem. Biodiversity 2019, 16, e1800673
[5] A. Peksel, C. Celik, N. Ocal, R. Yanardag, ‘Antioxidant and
Results of antioxidant activity were expressed as
EC50 values [as μg sample per ml], defined as the
amount of antioxidant necessary to decrease the initial
Radical Scavenging Activities of Some Norcantharidin and
Bridged Perhydroisoindole Derivatives’, J. Serb. Chem. Soc.
2013, 78, 15–25.
*
ABTS+ concentration by 50% and also as a Trolox
[6] A. Ariffin, N. A. Rahman, W. A. Yehye, A. A. Alhadi, F. A.
Kadir, ‘PASS-Assisted Design, Synthesis and Antioxidant
Evaluation of New Butylated Hydroxytoluene Derivatives’,
Eur. J. Med. Chem. 2014, 87, 564–577.
equivalent antioxidant capacity (TEAC, [mM of Trolox
per g sample]) based on their EC50 values.
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Metal chelating power (CHEL) was determined by
the method of Guo et al.[33] Subsequently, the absorb-
ance of the solution was recorded spectrophotometri-
cally at 562 nm. The percentage of inhibition of
ferrozine-Fe2+ complex formation was calculated
according to the following formula:
% inhibition ¼ ½1À ðAp=AcÞ� � 100
[10] C. Chang, Y. Zhu, X. Tang, W. Tao, ‘The Anti-Proliferative
Effects of Norcantharidin on Human HepG2 Cells in Cell
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J.-P. Lin, J.-G. Chung, ‘Norcantharidin Triggers Cell Death
and DNA Damage through S-Phase Arrest and ROS-
Modulated Apoptotic Pathways in TSGH 8301 Human
Urinary Bladder Carcinoma Cells’, Int. J. Oncol. 2012, 41,
1050–1060.
where Ac – the absorbance of control and Ap – the
absorbance in the presence of sample.
Results were expressed as % inhibition of sample
for concentration of 5 mg of sample per ml.
[12] B. Shen, P.-J. He, C.-L. Shao, ‘Norcantharidin Induced
DU145 Cell Apoptosis through ROS-Mediated Mitochon-
drial Dysfunction and Energy Depletion’, PLoS One 2013, 8,
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[13] C.-B. Yeh, M.-J. Hsieh, Y.-H. Hsieh, M.-H. Chien, H.-L. Chiou,
S.-F. Yang, ‘Antimetastatic Effects of Norcantharidin on
Hepatocellular Carcinoma by Transcriptional Inhibition of
MMP-9 through Modulation of NF-κB Activity’, PLoS One
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J.-Q. Zhang, ‘Norcantharidin Enhances Bortezomib-Anti-
myeloma Activity in Multiple Myeloma Cells In Vitro and in
Nude Mouse Xenografts’, Leuk. Lymphoma 2013, 54, 607–
618.
Author Contribution Statement
Anna Pachuta-Stec designed the study, performed the
synthesis of new norcantharidin analogs, analyzed the
spectral data and wrote the first draft of the manu-
script excluding the antiradical activity analysis. Renata
Nowak supervised the antiradical activity tests and
wrote the antiradical activity section of this manu-
script. Wioleta Pietrzak performed the antiradical
activity analysis of synthesized compounds. Monika
Pitucha has made revision of the article. All authors
read and approved the final manuscript.
[15] J.-Y. Wu, C.-D. Kuo, C.-Y. Chu, M.-S. Chen, J.-H. Lin, Y.-J.
Chen, H.-F. Liao, ‘Synthesis of Novel Lipophilic N-Substi-
tuted Norcantharimide Derivatives and Evaluation of Their
Anticancer Activities’, Molecules 2014, 19, 6911–6928.
[16] M. Tarleton, J. Gilbert, J. A. Sakoff, A. McCluskey, ‘Synthesis
and Anticancer Activity of a Series of Norcantharidin
Analogues’, Eur. J. Med. Chem. 2012, 54, 573–581.
[17] A. Pachuta-Stec, A. Szuster-Ciesielska, ‘New Norcantharidin
Analogues: Synthesis and Anticancer Activity’, Arch. Pharm.
Chem. Life Sci. 2015, 348, 1–11.
[18] A. McCluskey, M. C. Bowyer, E. Collins, A. T. R. Sim, J. A.
Sakoff, M. L. Baldwin, ‘Anhydride Modified Cantharidin
Analogues: Synthesis, Inhibition of Protein Phosphatases 1
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2000, 10, 1687–1690.
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Received December 19, 2018
Accepted February 7, 2019
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