Synthesis and Biological Evaluation of (+)-Usnic Acid Derivatives as Potential Anti- Toxoplasma gondii Agents

Article abstract of DOI:10.1021/acs.jafc.9b02173

Six series of (+)-usnic acid derivatives were synthesized. The IC₅₀ values of these compounds were determined in T. gondii infected HeLa cells (μM) and in HeLa cells (μM), and their selectivity indexes (SI) were calculated. In vitro, most of th

Full text of DOI:10.1021/acs.jafc.9b02173

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Functional Structure/Activity Relationships
Synthesis and biological evaluation of (+)-usnic acid
derivatives as potential anti-Toxoplasma gondii agents
yan hong Guo, mei chun Jin, ming hai Zhang, Mei Chun Jin, Kun Qing Shen, and Shan Zhe Quan
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02173 • Publication Date (Web): 31 Jul 2019
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Page 1 of 51
Journal of Agricultural and Food Chemistry
Synthesis and biological evaluation of (+)-usnic acid derivatives as potential
anti-Toxoplasma gondii agents
Hong-Yan Guo, ChunMei Jin, Hai-Ming Zhang, Chun-Mei Jin**, Qing-Kun Shen*, Zhe-Shan
Quan
Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain,
Affiliated Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin, 133002,
China.
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Abstract
Six series of (+)-usnic acid derivatives were synthesized. The IC values of these compounds
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were determined in T. gondii-infected HeLa cells (μM) and in HeLa cells (μM), and their selectivity
indexes (SI) were calculated. In vitro, most of the derivatives tested in this study exhibited more
anti-T. gondii activity than that of the parent compound (+)-usnic acid and the positive control
drugs.
Among
these
derivatives,
methyl(E)-
(1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(1H)-ylidene)
ethyl)phenylalaninate (D3) showed the most effective anti-T. gondii activity (Selectivity: > 2.77).
And compared with the clinically used positive control drugs sulfadiazine (Selectivity: 1.15),
pyrimethamine (Selectivity: 0.89), spiramycin (Selectivity: 0.72) and the lead compound (+)-usnic
acid (Selectivity: 0.96), D3 showed better results in vitro. Furthermore, D3 and
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(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-(quinolin-6-ylamino)ethylidene)dibenzo[b,d]furan-1,3
(2H,9bH)-dione (F3) had more inhibitory effects on T. gondii (inhibition rate: 76.0% and 64.6%) in
vivo than spiramycin (inhibition rate: 55.2%) and in the peritoneal cavity of mice, the number of
tachyzoites was significantly reduced (p < 0.001) in vivo. Additionally, some biochemical
parameters were measured, spleen indexes were comprehensively evaluated, and the results
indicated that mice treated with both compound D3 and compound F3 showed reduced
hepatotoxicity and significantly enhanced antioxidative effects compared to the normal group. And
granuloma and cyst-formation were effected by the inhibition of compound D3 and compound F3
in liver sections. Overall, these results indicated that D3 and F3 for use as anti-T. gondii agents are
promising lead compounds.
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Key words: Usnic acid, Derivatives, Toxoplasma gondii, Tachyzoite
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1. Introduction
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Toxoplasma gondii (T. gondii) can cause toxoplasmosis to affect the healthy of between
humans and animals [1,2]. The life cycle of T. gondii is complex, it can be differentiated from a
rapid replication form (tachyzoite) to a less metabolically active form (bradyzoite) encased in a
tissue cyst. Tachyzoites can spread through the placenta of pregnant women, by eating
contaminated water or food by parasitic oocysts transmitted by infected felines, by ingesting
uncooked meat with parasitic tissue cysts, thereby establish acute infections in humans and other
animals [3-5]. Toxoplasmosis is a zoonosis affecting a third of humankind [6-7]. However,
nowadays there is not any reliable drugs to prevent and treat toxoplasmosis due to different
biological characteristics, its multifarious pathogenesis, and the complication of the life cycle of T.
gondii. The current anti-T. gondii drugs have many defects, like failure in immuno-compromised
individuals, frequent recurrence, high toxicity and killing protozoa and oocysts is not thorough [8,9].
Over the past few years, in the market, the proportion of drugs based on natural products has
increased. In this background, natural products have proven useful as core sources of novel
compositions. Additionally, The found and development of anti-T. gondii drug make us known the
function of strong anti-T. gondii from natural products and their derivatives. [10,11]. Therefore, the
discovery of more effective and less toxic derivatives through structural modification of natural
products in the study of anti-T. gondii drugs is promising.
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Usnic acid (Figure 1) is produced by Alectoria (Alectoriaceae), Parmelia (Parmeliaceae),
Evernia, Ramalina (Ramalinaceae), Usnea (Usneaceae), Cladonia (Cladoniaceae), and other lichen
genera. It is the most well-known lichen secondary metabolite [12]. Many physiological and
biological activities can be found in usnic acid, like analgesic and anti-pyretic [13],
anti-antimicrobial [14], anti-inflammatory [15], anti-proliferative [16], anti-mitotic [17],
anti-parasitic [18]. Usnic acid is present as (+) and (-) enantiomers. Compared with (-) enantiomers,
the (+) enantiomeric activity is better, and it produces selective activity against Staphylococcus
mutants [19]. According to recent research reports, usnic acid has a lower concentration of
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inhibitory effects on Trichomonas vaginalis, Leishmania spp, and pathogenic protozoan in vitro
[12]. The excellent inhibitory effect of usnea sodium on T. gondii in vitro has been proved by
Chinese researchers [20]. But, the toxicity of usnic acid, especially liver failure[21]and cultured
mouse hepatocyte necrosis [22], has also been reported. Nevertheless, poor solubility in water [23]
and hepatotoxicity [24] limit the application of usnic acid to some extent. In addition, as we all
known, there is no research of usnic acid derivatives for anti-T. gondii. Therefore, in the present
study, we introduced different crucial fragments in (+)-usnic acid in order to obtain compounds
with low toxicity and high efficiency against T. gondii.
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Due to the effective biological and synthetic importance of triazole and its fused phenyl
derivatives, its chemical properties have obtained considerable attention. [25-27]. A series of
1,2,3-triazole-conjugated phenyl derivatives promote the latent antiparasitic drugs developing and
in the T. gondii model, five of these derivatives showed outstanding selectivity in vitro, according
to Sharling et al. [27]. The above studies show that triazole compounds have latent anti-T. gondii
activity. Amino acids as the fundamental functional and biological units to play a quite major role
in human metabolism and make up proteins. Some of biological functions were found in amino acid
derivatives, such as immunomodulatory [28], anti-parasitic [29-31], bactericidal [32],
anti-tuberculosis [33], anti-cancer [34,35] and so on. The combine of molecular structure of a drug
and an amino acid brings two advantages. For the one hand, the method could increase biological
activity and reduce toxic effects on cells [36-40]. For the another hand, the approach should
enhance bioavailability of drug due to increased solubility and the active transport [41,42]. In
usually, the number of polar groups for a compound would affect solubility, such as amide groups,
amino, carboxyl, and hydroxyl. The biological activity of a compound may change as its solubility
changes. In addition, an amide group can be used as a hydrogen donor, decreasing their sensibility
to enzymatic hydrolysis, and it can also improve the stability of compounds. Similarly, a drug with
a phosphonate introduced can display enhanced solubility and drug-like properties by regulating the
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distribution coefficient [43,44]. In addition, pyrazole and quinolines have been reported to possess
anti-T. gondii activity [45,46].
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The idea of designing and synthesizing six series of (+)-usnic acid derivatives was came out of
the combination principles of drugs and the findings above. The IC50 values of the target
compounds were determined in T. gondii-infected HeLa cells (μM) and HeLa cells (μM), and their
selectivity index (SI) was calculated. It is generally reflected the efficacy of a compound against T.
gondii and toxicity for host cells. [47]. Our synthesis and screening goal was to obtain compounds
with greater selectivity than the leading compound. Because testing in vivo is an important aspect in
assessing activity, the two most active anti-T. gondii derivatives were selected for animal studies.
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2. Experimental Section
2.1. General procedures
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The chemical reactions involved in this experiment were examined using thin-layer
chromatography (TLC). The determination of the melting point using an open capillary tube and
temperature was uncorrected. Deuterated chloroform (CDCl ) was used as a solvent for the
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determination of C-NMR and H-NMR spectra on BRUKER AV-300 (Bruker, Switzerland) with
ppm as a chemical shift unit. Thermo Scientific LTQ Orbitrap XL in ESI mode was used as the
measurement of high-resolution mass spectra. All analytical grade solvent and chemicals were used
directly without further processing and were obtained commerically.
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2.2. Representative Intermediates H-NMR Spectra
2.2.1. (1-phenyl-1H-1,2,3-triazol-4-yl)methanamine (1a)
1
H-NMR (CDCl , 300MHz, ppm): δ 7.91 (s, 1H, -N-CH=), 7.79-7.72 (m, 2H, Ar-OH),
3
1
00
7.59-7.42 (m, 3H, Ar-OH), 4.10 (s, 2H, =C-CH -), 1.71 (s, 2H, -CH -NH ).
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2.2.2. Diethyl (amino(phenyl)methyl)phosphonate (2a)
1
103
104
105
H-NMR (CDCl , 300MHz, ppm): δ 7.51-7.30 (m, 5H, Ar-H), 4.27 (d, J = 17.1 Hz, 1H,
3
Ar-CH-), 4.15-3.81 (m, 4H, -O-CH -), 1.88 (s, 2H, -CH-NH ), 1.24 (dt, 6H, J = 29.1 Hz, J = 7.2 Hz,
2
2
-CH -CH ).
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1
06
07
2.3. Procedure for the preparation of compound A1-A8
The (+)-usnic acid (172 mg,
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0.5
mmol),
and
different
(1-phenyl-1H-1,2,3-triazol-4-yl)methanamines (0.6 mmol) were stirred in 10 mL absolute ethanol at
78℃ for 2-3 h. The crude product was obtained under reduced pressure by evaporation of the
solvent after confirming completion of the reaction by TLC. The silica gel column chromatography
was used for products purification (silica gel, methanol/dichloromethane, 1:200-1:100 v/v). The
characteristics of the spectroscopic and physical data were as shown below.
1
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1
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2.3.1.
(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-(((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)amino)ethy
lidene)dibenzo[b,d]furan-1,3(2H,9bH)-dione (A1)
1
1
1
1
1
1
1
1
1
18
19
20
21
22
23
24
25
Yellow crystal. M.p. 129-130℃. Yield: 62%. H-NMR (CDCl , 300 MHz, ppm): δ 13.98 (brs, 1H,
3
Ar-OH), 13.58 (s, 1H, -C-NH-CH -), 11.86 (brs, 1H, Ar-OH), 8.02 (s, 1H, triazole-H), 7.74 (d, J =
2
7.5 Hz, 2H, Ar-H), 7.58-7.46 (m, 3H, Ar-H), 5.80 (s, 1H, O=C-CH=), 4.92-4.90 (m, 2H, -NH-CH -),
2
2.80 (s, 3H, CH -C=O), 2.67 (s, 3H, Ar-CH ), 2.10 (s, 3H, -NH-C-CH ), 1.71 (s, 3H, O=C-C-CH ).
3
3
3
3
1
3
C-NMR (CDCl , 75 MHz, ppm): δ 200.63, 198.47, 175.40 , 174.15, 163.44, 158.12, 155.76,
3
142.85, 136.62, 129.85 (2C), 129.19 (2C), 120.57 (2C), 120.26, 107.99, 104.90, 102.61, 102.37,
+
+
101.30, 57.18, 39.42, 31.94, 31.26, 18.63, 7.47. ESI-HRMS (m/z): calcd for C H N O [M+H] :
2
7
25
4
6
501.17686; found: 501.17672.
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2.3.2.
(E)-6-acetyl-2-(1-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethylidene)-7,9-dihydro
xy-8,9b-dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione (A2)
1
1
1
1
1
1
1
1
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36
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Yellow crystal. M.p. 135-136℃. Yield: 58%. H-NMR (CDCl , 300 MHz, ppm): δ 13.92 (brs, 1H,
3
Ar-OH), 13.35 (s, 1H, -C-NH-CH -), 11.81 (brs, 1H, Ar-OH), 8.07 (s, 1H, triazole-H), 7.69 (d, J =
2
8.7 Hz, 2H, Ar-H), 7.50 (d, J = 8.4 Hz, 2H, Ar-H), 5.76 (s, 1H, O=C-CH=), 4.90 (d, J = 5.1 Hz, 2H,
-NH-CH -), 2.78 (s, 3H, CH -C=O), 2.65 (s, 3H, Ar-CH ), 2.07 (s, 3H, -NH-C-CH ), 1.68 (s, 3H,
2
3
3
3
1
3
O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.67, 198.49, 190.23, 175.40, 174.16, 163.44,
3
3
158.08, 155.75, 143.14, 135.08, 135.00, 130.04 (2C), 121.71 (2C), 120.23, 107.98, 104.88, 102.62,
+
102.35, 101.30, 57.20, 39.39, 31.97, 31.29, 18.66, 7.51. ESI-HRMS (m/z): calcd for C H ClN O
6
2
7
24
4
+
[M+H] : 535.13789; found: 535.13745.
1
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1
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39
40
41
2.3.3.
(E)-6-acetyl-2-(1-(((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethylidene)-7,9-dihydro
xy-8,9b-dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione (A3)
1
1
1
1
1
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49
Yellow crystal. M.p. 137-138℃. Yield: 55%. H-NMR (CDCl , 300 MHz, ppm): δ 13.99 (brs, 1H,
3
Ar-OH), 13.36 (s, 1H, -C-NH-CH -), 11.89 (brs, 1H, Ar-OH), 8.04 (s, 1H, triazole-H), 7.67-7.59 (m,
2
2H, Ar-H), 7.53-7.48 (m, 2H, Ar-H), 5.80 (s, 1H, O=C-CH=), 4.93 (d, J = 5.7 Hz, 2H, -NH-CH -),
2
2.80 (s, 3H, CH -C=O), 2.67 (s, 3H, Ar-CH ), 2.10 (s, 3H, -NH-C-CH ), 1.72 (s, 3H, O=C-C-CH ).
3
3
3
3
1
3
C-NMR (CDCl , 75 MHz, ppm): δ 200.60, 198.45, 190.17, 175.40, 174.12, 163.40, 158.08,
3
155.73, 141.90, 134.41, 131.08, 130.78, 128.44, 128.02, 127.69, 124.10, 107.95, 104.88, 102.59,
+
102.34, 101.37, 57.14, 39.37, 31.90, 31.22, 18.63, 7.43. ESI-HRMS (m/z): calcd for C H ClN O
6
2
7
24
4
+
[M+H] : 535.13789; found: 535.13748.
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2.3.4.
(E)-6-acetyl-2-(1-(((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethylidene)-7,9-dihydro
xy-8,9b-dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione (A4)
1
1
1
1
1
1
1
1
1
54
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57
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Yellow crystal. M.p. 128-129℃. Yield: 73%. H-NMR (CDCl , 300 MHz, ppm): δ 13.97 (brs, 1H,
3
Ar-OH), 13.36 (s, 1H, -C-NH-CH -), 11.84 (brs, 1H, Ar-OH), 7.98 (s, 1H, triazole-H), 7.74-7.69 (m,
2
2H, Ar-H), 7.28-7.22 (m, 2H, Ar-H), 5.79 (s, 1H, O=C-CH=), 4.91-4.89 (m, 2H, -NH-CH -), 2.79
2
(s, 3H, CH -C=O), 2.67 (s, 3H, Ar-CH ), 2.10 (s, 3H, -NH-C-CH ), 1.71 (s, 3H, O=C-C-CH ).
3
3
3
3
1
3
C-NMR (CDCl , 75 MHz, ppm): δ 220.63, 198.48, 175.37, 174.14, 164.24, 163.43, 160.93,
3
158.09, 155.74, 142.99, 132.91, 132.87, 122.66, 122.54, 120.44, 117.00, 116.70, 107.98, 104.88,
+
102.33, 101.29, 57.24, 39.38, 31.93, 31.24, 18.61, 7.47. ESI-HRMS (m/z): calcd for C H FN O
6
2
7
24
4
+
[M+H] : 519.16744; found: 519.16785.
1
62
1
1
1
63
64
65
2.3.5.
(E)-6-acetyl-2-(1-(((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethylidene)-7,9-dihydro
xy-8,9b-dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione (A5)
1
1
1
1
1
1
1
1
1
66
67
68
69
70
71
72
73
Yellow crystal. M.p. 134-135℃. Yield: 47%. H-NMR (CDCl , 300 MHz, ppm): δ 13.98 (brs, 1H,
3
Ar-OH), 13.36 (s, 1H, -C-NH-CH -), 11.83 (brs, 1H, Ar-OH), 8.01 (s, 1H, triazole-H), 7.66 (q, J =
2
9.0 Hz, 4H, Ar-H), 5.79 (s, 1H, O=C-CH=), 4.91-4.89 (m, 2H, -NH-CH -), 2.79 (s, 3H, CH -C=O),
2
3
1
3
2.67 (s, 3H, Ar-CH ), 2.10 (s, 3H, -NH-C-CH ), 1.71 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75
3
3
3
3
MHz, ppm): δ 200.54, 198.58, 198.49, 175.37, 174.15, 163.49, 158.11, 155.76, 143.16, 135.62 (2C),
132.99, 122.88, 121.91 (2C), 120.07, 108.03, 104.88, 102.33, 101.33, 90.35, 57.19, 39.35, 31.90,
+
+
31.14, 18.50, 7.43. ESI-HRMS (m/z): calcd for C H BrN O [M+H] : 579.08737; found:
2
7
24
4
6
579.08765.
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75
76
77
2.3.6.
(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-(((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-y
l)methyl)amino)ethylidene)dibenzo[b,d]furan-1,3(2H,9bH)-dione (A6)
1
1
1
1
1
1
1
1
1
78
79
80
81
82
83
84
85
Yellow crystal. M.p. 159-160℃. Yield: 47%. H-NMR (CDCl , 300 MHz, ppm): δ 13.96 (brs, 1H,
3
Ar-OH), 13.35 (s, 1H, -C-NH-CH -), 11.80 (brs, 1H, Ar-OH), 8.03 (s, 1H, triazole-H), 7.73 (d, J =
2
7.5 Hz, 2H, Ar-H), 7.50 (s, 2H, Ar-H), 5.78 (s, 1H, O=C-CH=), 4.89 (d, J = 5.1 Hz, 2H, -NH-CH -),
2
2.78 (s, 3H, CH -C=O), 2.67 (s, 3H, Ar-CH ), 2.09 (s, 3H, -NH-C-CH ), 1.70 (s, 3H, O=C-C-CH ).
3
3
3
3
1
3
C-NMR (CDCl , 75 MHz, ppm): δ 200.67, 198.49, 197.11, 175.30, 163.39, 157.99, 155.69,
3
153.14, 143.14, 130.34, 126.20, 126.16, 125.94, 121.12, 120.97, 120.85, 120.30 (2C), 119.96,
108.00, 104.79, 101.27, 39.29, 31.89, 31.22, 18.58, 7.55, 7.47. ESI-HRMS (m/z): calcd for
+
+
C H F N O [M+H] : 569.16425; found: 569.16456.
2
8
24
3
4
6
1
86
1
1
1
87
88
89
2.3.7.
(E)-6-acetyl-7,9-dihydroxy-2-(1-(((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethylid
ene)-8,9b-dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione (A7)
1
1
1
1
1
1
1
1
1
90
91
92
93
94
95
96
97
Yellow crystal. M.p. 132-134℃. Yield: 53%. H-NMR (CDCl , 300 MHz, ppm): δ 13.97 (brs, 1H,
3
Ar-OH), 13.38 (s, 1H, -C-NH-CH -), 11.89 (brs, 1H, Ar-OH), 7.95 (s, 1H, triazole-H), 7.64 (d, J =
2
9.0 Hz, 2H, Ar-H), 7.05 (d, J = 9.0 Hz, 2H, Ar-H), 5.81 (s, 1H, O=C-CH=), 4.92-4.90 (m, 2H,
-NH-CH -), 3.90 (s, 3H, -OCH ), 2.81 (s, 3H, CH -C=O), 2.69 (s, 3H, Ar-CH ), 2.11 (s, 3H,
2
3
3
3
1
3
-NH-C-CH ), 1.73 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.55, 198.50,
3
3
3
175.37, 163.51, 160.16, 158.18, 155.80, 142.61, 130.11, 122.24 (2C), 120.33, 114.88 (2C), 108.05,
104.94, 102.47, 102.39, 102.34, 102.24, 101.35, 55.62, 39.47 (2C), 31.90, 31.14, 18.52, 7.42.
+
+
ESI-HRMS (m/z): calcd for C H N O [M+H] : 531.18743; found: 531.18777.
2
8
27
4
7
1
98
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2
2
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00
01
2.3.8.
(E)-6-acetyl-7,9-dihydroxy-2-(1-(((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethylid
ene)-8,9b-dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione (A8)
1
2
2
2
2
2
2
2
2
02
03
04
05
06
07
08
09
Yellow crystal. M.p. 129-130℃. Yield: 58%. H-NMR (CDCl , 300 MHz, ppm): δ 13.92 (brs, 1H,
3
Ar-OH), 13.36 (s, 1H, -C-NH-CH -), 11.92 (brs, 1H, Ar-OH), 8.17 (s, 1H, triazole-H), 7.80 (d, J =
2
8.1 Hz, 2H, Ar-H), 7.49-7.43 (m, 1H, Ar-H), 7.16-7.10 (m, 2H, Ar-H), 5.79 (s, 1H, O=C-CH=),
4.91-4.89 (m, 2H, -NH-CH -), 3.92 (s, 3H, -OCH ), 2.80 (s, 3H, CH -C=O), 2.67 (s, 3H, Ar-CH ),
2
3
3
3
1
3
2.10 (s, 3H, -NH-C-CH ), 1.72 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.65,
3
3
3
198.43, 190.13, 175.38, 174.07, 163.41, 158.14, 155.79, 150.88, 130.45, 125.81, 125.31, 124.32,
121.28, 112.32, 112.22, 107.93, 104.96, 102.58, 102.43, 101.29, 57.11, 56.00, 39.53, 31.95, 31.27,
+
+
18.71, 7.47. ESI-HRMS (m/z): calcd for C H N O [M+H] : 531.18743; found: 531.18776.
2
8
27
4
7
2
2
10
11
2.4. Procedure for the preparation of compound B1-B5
2
2
2
2
2
2
12
13
14
15
16
17
The (+)-usnic acid (172 mg, 0.5 mmol), and different 2-aminoethyl secondary amines (0.6
mmol) were stirred in 10 mL absolute ethanol at 78℃ for 2-3 h. The crude product was obtained
under reduced pressure by evaporation of the solvent after confirming completion of the reaction by
TLC. The silica gel column chromatography was used for products purification (silica gel,
methanol/dichloromethane, 1:200-1:100 v/v). The characteristics of the spectroscopic and physical
data were as shown below.
2
18
2
2
2
19
20
21
2.4.1.
(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-((2-(pyrrolidin-1-yl)ethyl)amino)ethylidene)dibenzo[
b,d]furan-1,3(2H,9bH)-dione (B1)
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2
2
2
2
2
2
2
2
22
23
24
25
26
27
28
29
Yellow crystal. M.p. 96-97℃. Yield: 82%. H-NMR (CDCl , 300 MHz, ppm): δ 13.36 (brs, 1H,
3
Ar-OH), 12.04 (brs, 1H, Ar-OH), 5.78 (s, 1H, O=C-CH=), 3.62-3.56 (m, 2H, -NH-CH -), 2.84-2.80
2
(m, 2H, -NH-CH -CH -), 2.67 (s, 3H, CH -C=O), 2.64 (s, 3H, Ar-CH ), 2.62-2.56 (m, 4H,
2
2
3
3
pyrrole-H), 2.09 (s, 3H, -NH-C-CH ), 1.92-1.80 (m, 4H, pyrrole-H), 1.70 (s, 3H, O=C-C-CH ), 1.25
3
3
1
3
(s, 1H, -C-NH-CH -). C-NMR (CDCl , 75 MHz, ppm): δ 200.57, 198.11, 174.51, 173.86, 163.47,
2
3
158.36 (2C), 155.91, 107.91, 105.16, 102.54 (2C), 101.35, 56.94, 54.03 (2C), 53.96, 43.29, 31.94,
+
+
31.14, 23.71 (2C), 18.55, 7.41. ESI-HRMS (m/z): calcd for C H N O [M+H] : 441.20201;
2
4
29
2
6
found: 441.20211.
2
30
2
2
2
31
32
33
2.4.2.
(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-((2-(piperidin-1-yl)ethyl)amino)ethylidene)dibenzo[
b,d]furan-1,3(2H,9bH)-dione (B2)
1
2
2
2
2
2
2
2
2
34
35
36
37
38
39
40
41
Yellow crystal. M.p. 98-99℃. Yield: 87%. H-NMR (CDCl , 300 MHz, ppm): δ 13.37 (brs, 1H,
3
Ar-OH), 12.06 (brs, 1H, Ar-OH), 5.79 (s, 1H, O=C-CH=), 3.58-3.52 (m, 2H, -NH-CH -), 2.68 (s,
2
3H, CH -C=O), 2.66-2.62 (m, 5H, Ar-CH -NH-CH -CH -), 2.49-2.46 (m, 4H, piperidine-H), 2.10
3
3,
2
2
(s, 3H, -NH-C-CH ), 1.71 (s, 3H, O=C-C-CH ), 1.68-1.61 (m, 4H, piperidine-H), 1.51-1.46 (m, 2H,
3
3
1
3
piperidine-H), 1.26 (s, 1H, -C-NH-CH -). C-NMR (CDCl , 75 MHz, ppm): δ 220.53 (2C), 197.99,
2
3
174.29, 173.70, 163.41, 158.35, 155.89, 107.83, 105.16, 102.59 (2C), 101.29, 56.46, 54.44 (2C),
+
41.59, 31.90, 31.09, 25.90 (3C), 24.20, 18.63, 7.37. ESI-HRMS (m/z): calcd for C H N O
6
2
5
31
2
+
[M+H] : 455.21766; found: 455.21793.
2
42
2
2
2
43
44
45
2.4.3.
(E)-6-acetyl-2-(1-((2-(dimethylamino)ethyl)amino)ethylidene)-7,9-dihydroxy-8,9b-dimethyldibenzo[
b,d]furan-1,3(2H,9bH)-dione (B3)
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1
2
2
2
2
2
2
2
46
47
48
49
50
51
52
Yellow crystal. M.p. 83-84℃. Yield: 84%. H-NMR (CDCl , 300 MHz, ppm): δ 13.37 (brs, 1H,
3
Ar-OH), 12.05 (brs, 1H, Ar-OH), 5.79 (s, 1H, O=C-CH=), 3.58-3.49 (m, 2H, -NH-CH -), 2.68 (s,
2
3H, CH -C=O), 2.65 (s, 3H, Ar-CH ), 2.64-2.62 (m, 2H, -NH-CH -CH -), 2.35 (s, 6H, -N-CH ),
3
3
2
2
3
1
3
2.10 (s, 3H, -NH-C-CH ), 1.71 (s, 3H, O=C-C-CH ), 1.26 (s, 1H, -C-NH-CH -). C-NMR (CDCl ,
3
3
2
3
75 MHz, ppm): δ 220.69 (2C), 198.15, 174.39, 173.90, 163.40, 158.31, 155.89, 114.27, 107.83,
105.16, 102.50, 101.31, 57.11 (2C), 45.32 (2C), 41.99, 31.99, 31.30, 18.79, 7.48. ESI-HRMS (m/z):
+
+
calcd for C H N O [M+H] : 415.18636; found: 415.18656.
2
2
27
2
6
2
53
2
2
2
54
55
56
2.4.4.
(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-((2-(4-methylpiperazin-1-yl)ethyl)amino)ethylidene)
dibenzo[b,d]furan-1,3(2H,9bH)-dione (B4)
1
2
2
2
2
2
2
2
57
58
59
60
61
62
63
Yellow crystal. M.p. 80-82℃. Yield: 72%. H-NMR (CDCl , 300 MHz, ppm): δ 13.35 (brs, 1H,
3
Ar-OH), 12.03 (brs, 1H, Ar-OH), 5.77 (s, 1H, O=C-CH=), 3.58-3.53 (m, 2H, -NH-CH -), 2.72-2.59
2
(m, 16H, -NH-CH -CH -, CH -C=O, -NH-C-CH , piperazine-H), 2.35 (s, 3H, -N-CH ), 2.08 (s, 3H,
2
2
3
3
3
1
3
Ar-CH ), 1.69 (s, 3H, O=C-C-CH ), 1.24 (s, 1H, -C-NH-CH -). C-NMR (CDCl , 75 MHz, ppm):
3
3
2
3
δ 200.64 (2C), 197.99, 174.32, 173.75, 163.37, 158.28, 155.87, 107.81, 105.11, 102.61, 102.56,
101.28, 55.48, 54.85 (2C), 52.63 (2C), 45.75, 41.19, 31.95, 31.89, 31.23, 18.81, 7.43. ESI-HRMS
+
+
(m/z): calcd for C H N O [M+H] : 470.22856; found: 470.22876.
2
5
32
3
6
2
64
2
2
2
65
66
67
2.4.5.
(E)-6-acetyl-2-(1-((2-(diethylamino)ethyl)amino)ethylidene)-7,9-dihydroxy-8,9b-dimethyldibenzo[b,
d]furan-1,3(2H,9bH)-dione (B5)
1
2
2
2
68
69
70
Yellow crystal. M.p. 106-107℃. Yield: 96%. H-NMR (CDCl , 300 MHz, ppm): δ 13.36 (brs, 1H,
3
Ar-OH), 12.08 (brs, 1H, Ar-OH), 5.78 (s, 1H, O=C-CH=), 3.55-3.49 (m, 2H, -NH-CH -), 2.77-2.73
2
(m, 2H, -NH-CH -CH -), 2.67 (s, 3H, CH -C=O), 2.64 (s, 3H, Ar-CH ), 2.62-2.58 (m, 4H,
2
2
3
3
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2
2
2
2
71
72
73
74
75
-N-CH -CH ), 2.08 (s, 3H, -NH-C-CH ), 1.69 (s, 3H, O=C-C-CH ), 1.25 (s, 1H, -C-NH-CH -), 1.07
2 3 3 3 2
1
3
(t, 6H, J = 7.2, -N-CH -CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.58 (2C), 198.05, 174.35,
2
3
3
173.74, 163.46, 158.39, 155.94, 107.87, 105.20, 102.62, 102.41, 101.34, 56.86, 51.15, 47.01 (2C),
+
+
42.62, 31.95, 31.14, 18.69, 11.82 (2C), 7.41. ESI-HRMS (m/z): calcd for C H N O [M+H] :
2
4
31
2
6
443.21766; found: 443.21754.
2
2
76
77
2.5. Procedure for the preparation of compound C1-C5
2
2
2
2
2
2
78
79
80
81
82
83
The
(+)-usnic
acid
(172
mg,
0.5
mmol),
and
different
diethyl
(amino(phenyl)methyl)phosphonates (0.6 mmol) was stirred in 10 mL absolute ethanol at 78℃ for
2-3 h. The crude product was obtained under reduced pressure by evaporation of the solvent after
confirming completion of the reaction by TLC. The silica gel column chromatography was used for
products purification (silica gel, methanol/dichloromethane, 1:200-1:100 v/v). The characteristics of
the spectroscopic and physical data were as shown below.
2
84
2
2
2
85
86
87
2.5.1.
Diethyl(E)-(((1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(
1H)-ylidene)ethyl)amino)(phenyl)methyl)phosphonate (C1)
1
2
2
2
2
2
2
2
2
88
89
90
91
92
93
94
95
Yellow crystal. M.p. 98-99℃. Yield: 50%. H-NMR (CDCl , 300 MHz, ppm): δ 14.64 (brs, 1H,
3
Ar-OH), 13.35 (s, 1H, -C-NH-CH -), 11.78 (d, J = 3.3 Hz, 1H, Ar-OH), 7.47-7.41 (m, 5H, Ar-H),
2
5.86 (d, J = 5.7 Hz, 1H, O=C-CH=), 5.23-5.12 (m, 1H, -NH-CH-), 4.12-3.98 (m, 4H, -O-CH -),
2
3.49 (s, 3H, CH -C=O), 2.69 (s, 3H, Ar-CH ), 2.12-2.09 (m, 3H, -NH-C-CH ), 1.77-1.69 (m, 3H,
3
3
3
1
3
O=C-C-CH ), 1.31-1.22 (m, 6H, -CH -CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.60, 198.57,
3
2
3
3
190.90, 174.52, 174.46, 163.47 (2C), 158.13, 132.77, 129.15, 128.91, 127.71, 127.65, 127.58,
108.08, 104.89, 102.26, 102.16, 101.32, 64.04, 57.16, 55.12, 31.84, 31.28 (2C), 18.38, 16.38, 16.31,
+
+
7.45. ESI-HRMS (m/z): calcd for C H NO P [M+H] : 570.18874; found: 570.18855.
2
9
33
9
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2
96
2
2
2
97
98
99
2.5.2.
Diethyl(E)-(((1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(
1H)-ylidene)ethyl)amino)(4-chlorophenyl)methyl)phosphonate (C2)
1
3
3
3
3
3
3
3
3
3
00
01
02
03
04
05
06
07
08
Yellow crystal. M.p. 120-121℃. Yield: 64%. H-NMR (CDCl , 300 MHz, ppm): δ 14.65 (brs, 1H,
3
Ar-OH), 13.36 (s, 1H, -C-NH-CH -), 11.71 (d, J = 3.6 Hz, 1H, Ar-OH), 7.42-7.38 (m, 4H, Ar-H),
2
5.86 (d, J = 6.0 Hz, 1H, O=C-CH=), 5.20-5.08 (m, 1H, -NH-CH-), 4.15-4.03 (m, 4H, -O-CH -),
2
2.69 (s, 3H, CH -C=O), 2.59 (s, 3H, Ar-CH ), 2.10-2.09 (m, 3H, -NH-C-CH ), 1.71 (d, J = 10.0 Hz,
3
3
3
1
3
3H, O=C-C-CH ), 1.33-1.25 (m, 6H, -CH -CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.48,
3
2
3
3
198.71, 198.64, 190.91, 174.57, 163.54, 158.10, 155.68, 134.98, 131.55, 129.32, 128.99, 128.93,
128.86, 108.18, 104.80, 103.05, 102.20, 101.35, 64.07, 57.55, 56.55, 54.54, 31.77, 31.14, 18.23,
+
+
16.34, 16.27, 7.38. ESI-HRMS (m/z): calcd for C H ClNO P [M+H] : 604.14977; found:
2
9
32
9
604.14996.
3
09
3
3
3
10
11
12
2.5.3.
Diethyl(E)-(((1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(
1H)-ylidene)ethyl)amino)(3,4-dichlorophenyl)methyl)phosphonate (C3)
1
3
3
3
3
3
3
3
13
14
15
16
17
18
19
Yellow crystal. M.p. 101-102℃. Yield: 66%. H-NMR (CDCl , 300 MHz, ppm): δ 14.70-14.65 (m,
3
1H, Ar-OH), 13.36 (s, 1H, -C-NH-CH -), 11.66 (d, J = 2.1 Hz, 1H, Ar-OH), 7.56-7.49 (m, 2H,
2
Ar-H), 7.37-7.31 (m, 1H, Ar-H), 5.88 (d, J = 4.6 Hz, 1H, O=C-CH=), 5.19-5.06 (m, 1H, -NH-CH-),
4.22-4.09 (m, 4H, -O-CH -), 2.69 (s, 3H, CH -C=O), 2.60 (s, 3H, Ar-CH ), 2.10 (s, 3H,
2
3
3
1
3
-NH-C-CH ), 1.77-1.71 (m, 3H, O=C-C-CH ), 1.37-1.30 (m, 6H, -CH -CH ). C-NMR (CDCl , 75
3
3
2
3
3
MHz, ppm): δ 200.63, 198.85, 198.78, 191.14, 191.09, 174.81, 163.61, 158.11, 155.75, 133.44,
131.16, 129.63, 126.84, 108.30, 104.83, 103.26, 103.21, 102.27, 102.19, 101.41, 64.32, 57.68,
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+
3
3
20
21
56.16, 54.12, 31.86, 31.31, 18.38, 16.37, 7.49. ESI-HRMS (m/z): calcd for C H Cl NO P
29 31 2 9
+
[M+H] : 638.11080; found: 638.11063.
3
22
3
3
3
23
24
25
2.5.4.
Diethyl(E)-(((1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(
1H)-ylidene)ethyl)amino)(p-tolyl)methyl)phosphonate (C4)
1
3
3
3
3
3
3
3
3
3
26
27
28
29
30
31
32
33
34
Yellow crystal. M.p. 106-107℃. Yield: 61%. H-NMR (CDCl , 300 MHz, ppm): δ 14.61 (brs, 1H,
3
Ar-OH), 13.36 (s, 1H, -C-NH-CH -), 11.81 (d, J = 5.1 Hz, 1H, Ar-OH), 7.38-7.30 (m, 2H, Ar-H),
2
7.23-7.20 (m, 1H, Ar-H), 5.85 (d, J = 6.0 Hz, 1H, O=C-CH=), 5.19-5.07 (m, 1H, -NH-CH-),
4.13-3.99 (m, 4H, -O-CH -), 2.69 (s, 3H, CH -C=O), 2.60 (s, 3H, Ar-CH ), 2.36 (d, J = 5.4 Hz, 3H,
2
3
3
Ar-CH ), 2.10 (s, 3H, -NH-C-CH ), 1.73-1.69 (m, 3H, O=C-C-CH ), 1.30-1.23 (m, 6H, -CH -CH ).
3
3
3
2
3
1
3
C-NMR (CDCl , 75 MHz, ppm): δ 200.61, 198.52, 190.86, 174.47, 163.46, 158.16, 138.90,
3
129.83 (2C), 127.61, 127.54 (2C), 127.47, 108.07, 104.92, 102.26, 102.16, 101.31, 90.33, 63.98,
63.80, 56.93, 54.89, 31.84, 31.67, 31.28, 21.14, 18.38, 16.33, 7.45. ESI-HRMS (m/z): calcd for
+
+
C H NO P [M+H] : 584.20439; found: 584.20471.
3
0
35
9
3
35
3
3
3
36
37
38
2.5.5.
Diethyl(E)-(((1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(
1H)-ylidene)ethyl)amino)(4-methoxyphenyl)methyl)phosphonate (C5)
1
3
3
3
3
3
3
39
40
41
42
43
44
Yellow crystal. M.p. 128-129℃. Yield: 51%. H-NMR (CDCl , 300 MHz, ppm): δ 14.58 (brs, 1H,
3
Ar-OH), 13.36 (s, 1H, -C-NH-CH -), 11.81 (d, J = 3.9 Hz, 1H, Ar-OH), 7.42-7.34 (m, 2H, Ar-H),
2
6.98-6.92 (m, 2H, Ar-H), 5.85 (d, J = 6.3 Hz, 1H, O=C-CH=), 5.17-5.06 (m, 1H, -NH-CH-),
4.09-4.04 (m, 4H, -O-CH -), 3.82 (d, J = 4.5 Hz, 3H, -O-CH ), 2.69 (s, 3H, -NH-C-CH ), 2.61 (s,
2
3
3
3H, CH -C=O), 2.10 (s, 3H, Ar-CH ), 1.73-1.69 (m, 3H, O=C-C-CH ), 1.31-1.22 (m, 6H,
3
3
3
1
3
-CH -CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.56, 198.58, 190.85, 163.65 (2C), 160.05,
2
3
3
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3
3
3
45
46
47
158.22, 155.82, 128.96 (2C), 128.89, 124.69, 114.63 (2C), 108.15, 104.95, 103.00, 102.27, 101.38,
64.02, 63.90, 56.67, 55.32, 54.60, 31.82, 31.18, 18.28, 16.40, 16.33, 7.42. ESI-HRMS (m/z): calcd
+
+
for C H NO P [M+H] : 600.19931; found: 600.19914.
3
0
35
10
3
48
49
3
2.6. Procedure for the preparation of compound D1-D4
3
3
3
3
3
50
51
52
53
54
The (+)-usnic acid (172 mg, 0.5 mmol), Et N (1.0 mmol), and different L-amino acid methyl
3
ester hydrochlorides (0.6 mmol) was stirred in 10 mL ethanol and 2 mL H O at 78℃ for 3-5 h.
2
The mixture was obtained under reduced pressure by evaporation of the solvent after confirming
completion of the reaction by TLC. Next, the mixture was poured into water (20 mL), extracted by
dichloromethane (15×3 mL), and the organic phase was washed with saturated NaCl solution (50
3
3
3
3
55
56
57
58
mL) and dried by sodium sulfate. The sodium sulfate was removed by filtration. The crude product
was obtained under reduced pressure by evaporation of the solvent. The silica gel column
chromatography was used for Products purification (silica gel, methanol/dichloromethane,
1:400-1:200 v/v). The characteristics of the spectroscopic and physical data were as shown below.
3
59
3
3
3
60
61
62
2.6.1.
Methyl(E)-(1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(1
H)-ylidene)ethyl)glycinate (D1)
1
3
3
3
3
3
63
64
65
66
67
Yellow crystal. M.p. 141-142℃. Yield: 57%. H-NMR (CDCl , 300 MHz, ppm): δ 13.89 (brs, 1H,
3
Ar-OH), 13.37 (s, 1H, -C-NH-CH -), 11.83 (brs, 1H, Ar-OH), 5.83 (s, 1H, O=C-CH=), 4.29-4.27
2
(m, 2H, -NH-CH -), 3.87 (s, 3H, -O-CH ), 2.68 (s, 3H, CH -C=O), 2.60 (s, 3H, Ar-CH ), 2.10 (s,
2
3
3
3
1
3
3H, -NH-C-CH ), 1.72 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.62, 200.55,
3
3
3
198.62, 175.04, 174.28, 167.45, 163.51, 158.13, 155.78, 108.04, 104.88, 102.25, 102.21, 101.35,
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+
3
3
68
69
62.40, 52.98, 45.02, 31.11, 18.60, 14.03, 7.36. ESI-HRMS (m/z): calcd for C H NO [M+H] :
21 22 8
416.13399; found: 416.13364.
3
70
3
3
3
71
72
73
2.6.2.
Methyl(E)-(1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(1
H)-ylidene)ethyl)tryptophanate (D2)
1
3
3
3
3
3
3
3
3
3
74
75
76
77
78
79
80
81
82
Yellow crystal. M.p. 253-254℃. Yield: 48%. H-NMR (CDCl , 300 MHz, ppm): δ 14.04 (brs, 1H,
3
Ar-OH), 13.38 (s, 1H, -C-NH-CH -), 11.86 (brs, 1H, Ar-OH), 8.17 (s, 1H, indole-NH), 7.58 (d, J =
2
7.2 Hz, 1H, indole-H), 7.39 (d, J = 7.8 Hz, 1H, indole-H), 7.26-7.14 (m, 3H, indole-H), 5.82 (s, 1H,
O=C-CH=), 4.85-4.74 (m, 1H, -NH-CH-), 3.84 (s, 3H, -O-CH ), 3.59-3.50 (m, 2H, -NH-CH-CH -),
3
2
2.70 (s, 3H, CH -C=O), 2.23 (s, 3H, Ar-CH ), 2.10 (s, 3H, -NH-C-CH ), 1.69 (s, 3H, O=C-C-CH ).
3
3
3
3
1
3
C-NMR (CDCl , 75 MHz, ppm): δ 200.65, 198.45, 198.35, 174.59, 170.06, 163.48 (2C), 158.16,
3
155.77, 136.19, 126.70, 123.72, 122.55, 119.96, 118.02, 117.87, 111.53, 108.65, 108.03, 104.96,
102.23, 101.33, 62.35, 57.39, 53.04, 31.75, 31.13, 29.42, 18.37, 7.38. ESI-HRMS (m/z): calcd for
+
+
C H N O [M+H] : 545.19184; found: 545.19165.
3
0
29
2
8
3
83
3
3
3
84
85
86
2.6.3.
Methyl(E)-(1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(1
H)-ylidene)ethyl)phenylalaninate (D3)
1
3
3
3
3
3
3
87
88
89
90
91
92
Yellow crystal. M.p. 94-95℃. Yield:50%. H-NMR (CDCl , 300 MHz, ppm): δ 14.08 (d, J = 7.8
3
Hz, 1H, Ar-OH), 13.35 (s, 1H, -C-NH-CH -), 11.80 (brs, 1H, Ar-OH), 7.31-7.29 (m, 3H, Ar-H),
2
7.19-7.16 (m, 2H, Ar-H), 5.30 (s, 1H, O=C-CH=), 4.68-4.66 (m, 1H, -NH-CH-), 3.84 (s, 3H,
-O-CH ), 3.38-3.32 (m, 1H, -NH-CH-CH -), 3.14-3.06 (m, 1H, -NH-CH-CH -), 2.68 (s, 3H,
3
2
2
1
3
CH -C=O), 2.24 (s, 3H, Ar-CH ), 2.09 (s, 3H, -NH-C-CH ), 1.68 (s, 3H, O=C-C-CH ). C-NMR
3
3
3
3
(CDCl , 75 MHz, ppm): δ 200.59, 198.52, 198.43, 174.62, 174.56, 169.63, 163.41, 158.08, 155.74,
3
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3
3
93
94
95
134.58, 129.18 (2C), 128.93 (2C), 127.75, 107.97, 104.90, 102.37, 102.32, 101.28, 62.41, 58.32,
53.09, 39.46, 31.77, 31.22, 18.19, 7.41. ESI-HRMS (m/z): calcd for C H NO [M+H]+:
+
2
8
28
8
506.18094; found: 506.18137.
3
96
3
3
3
97
98
99
2.6.4.
Methyl(E)-(1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]furan-2(1
H)-ylidene)ethyl)tyrosinate (D4)
1
4
4
4
4
4
4
4
4
4
00
01
02
03
04
05
06
07
08
Yellow crystal. M.p. 155-156℃. Yield: 52%. H-NMR (CDCl , 300 MHz, ppm): δ 13.83 (brs, 1H,
3
Ar-OH), 13.34 (s, 1H, -C-NH-CH -), 11.76 (brs, 1H, Ar-OH), 7.07-7.00 (m, 2H, Ar-H), 6.79-6.75
2
(m, 2H, Ar-H), 5.83 (s, 1H, O=C-CH=), 4.73-4.62 (m, 1H, -NH-CH-), 3.83-3.81 (m, 3H, -O-CH3),
3.29-3.22 (m, 1H, -NH-CH-CH -), 3.13-3.05 (m, 1H, -NH-CH-CH -), 2.66-2.65 (m, 3H, CH -C=O),
2
2
3
1
3
2.37 (s, 3H, Ar-CH ), 2.08 (s, 3H, -NH-C-CH ), 1.68 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75
3
3
3
3
MHz, ppm): δ 200.72, 200.65, 198.65, 174.80, 174.64, 169.72, 169.27, 163.52, 158.13, 155.76,
130.56, 130.49, 130.44, 126.08, 115.95, 115.91, 108.17, 104.92, 102.12, 101.38, 62.45, 58.50,
53.07, 38.51, 31.15, 18.46, 14.07, 7.42. ESI-HRMS (m/z): calcd for C H NO [M+H]+:
+
2
8
28
9
522.17586; found: 522.17633.
4
4
09
10
2.7. Procedure for the preparation of compound E1-E4
4
4
4
4
4
11
12
13
14
15
The (+)-usnic acid (172 mg, 0.5 mmol), Et N (1.0 mmol), and different phenylhydrazine
3
hydrochlorides (0.6 mmol) was stirred in 10 mL absolute ethanol at 78℃ for 3-5 h. The mixture
was obtained under reduced pressure by evaporation of the solvent after confirming completion of
the reaction by TLC. Next, the mixture was poured into water (20 mL), extracted by
dichloromethane (15×3 mL), and the organic phase was washed with saturated NaCl solution (50
4
16
mL) and dried by dry Na SO . Filtering to remove Na SO . The crude product was obtained under
2
4
2
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reduced pressure by evaporation of the solvent. The silica gel column chromatography was used for
products purification (silica gel, ethyl acetate/petroleum ether, 1:25-1:20 v/v). The characteristics of
the spectroscopic and physical data were as shown below.
4
20
4
4
4
21
22
23
2.7.1.
8-acetyl-5,7-dihydroxy-3,4a,6-trimethyl-1-phenyl-1,4a-dihydro-4H-benzofuro[3,2-f]indazol-4-one
(E1)
1
4
4
4
4
4
4
24
25
26
27
28
29
Yellow crystal. M.p. 179-180℃. Yield: 70%. H-NMR (CDCl , 300 MHz, ppm): δ 13.32 (s, 1H,
3
Ar-OH), 11.12 (s, 1H, Ar-OH), 7.58-7.45 (m, 5H, Ar-H), 6.25 (s, 1H, O=C-CH=), 2.64 (s, 3H,
1
3
CH -C=O), 2.59 (s, 3H, -N=C-CH ), 2.12 (s, 3H, Ar-CH ), 1.82 (s, 3H, O=C-C-CH ). C-NMR
3
3
3
3
(CDCl , 75 MHz, ppm): δ 200.35, 196.16, 172.93, 163.53, 157.61, 156.24, 151.42, 148.18, 137.85,
3
130.87, 129.65, 128.78, 128.73, 123.89, 110.67, 108.24, 103.96, 101.49, 89.27, 60.31, 31.19, 30.43,
+
+
13.26, 7.45. ESI-HRMS (m/z): calcd for C H N O [M+H] : 417.14450; found: 417.14472.
2
4
21
2
5
4
30
4
4
4
31
32
33
2.7.2.
8-acetyl-1-(3-chlorophenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1,4a-dihydro-4H-benzofuro[3,2-f]indaz
ol-4-one (E2)
1
4
4
4
4
4
4
4
34
35
36
37
38
39
40
Yellow crystal. M.p. 128-129℃. Yield: 47%. H-NMR (CDCl , 300 MHz, ppm): δ 13.33 (s, 1H,
3
Ar-OH), 11.02 (s, 1H, Ar-OH), 7.62 (s, 1H, Ar-H), 7.54-7.43 (m, 3H, Ar-H), 6.25 (s, 1H,
O=C-CH=), 2.66 (s, 3H, CH -C=O), 2.58 (s, 3H, -N=C-CH ), 2.12 (s, 3H, Ar-CH ), 1.82 (s, 3H,
3
3
3
1
3
O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.36, 196.15, 173.43, 163.59, 157.52, 156.17,
3
3
151.75, 148.34, 138.88, 135.46, 130.65, 128.79, 124.05, 121.81, 110.85, 108.39, 103.85, 101.52,
88.98, 60.47, 31.24, 30.43, 13.23, 7.46. ESI-HRMS (m/z): calcd for C H ClN O [M+H]+:
+
2
4
20
2
5
451.10553; found: 451.10593.
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41
4
4
4
42
43
44
2.7.3.
8-acetyl-1-(3-fluorophenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1,4a-dihydro-4H-benzofuro[3,2-f]indaz
ol-4-one (E3)
1
4
4
4
4
4
4
4
45
46
47
48
49
50
51
Yellow crystal. M.p. 166-167℃. Yield: 44%. H-NMR (CDCl , 300 MHz, ppm): δ 13.32 (s, 1H,
3
Ar-OH), 11.03 (s, 1H, Ar-OH), 7.54-7.50 (m, 1H, Ar-H), 7.40-7.34 (m, 2H, Ar-H), 7.22-7.16 (m,
1H, Ar-H), 6.28 (s, 1H, O=C-CH=), 2.66 (s, 3H, CH -C=O), 2.58 (s, 3H, -N=C-CH ), 2.12 (s, 3H,
3
3
1
3
Ar-CH ), 1.82 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.34, 196.15, 173.39,
3
3
3
164.59, 163.57, 157.52, 156.17, 151.70, 148.32, 131.04, 119.22, 115.81, 115.53, 111.66, 111.33,
110.86, 108.37, 101.51, 89.05, 60.46, 31.21, 30.42, 13.22, 7.45. ESI-HRMS (m/z): calcd for
+
+
C H FN O [M+H] : 435.13508; found: 435.13555.
2
4
20
2
5
4
52
4
4
4
53
54
55
2.7.4.
8-acetyl-5,7-dihydroxy-3,4a,6-trimethyl-1-(p-tolyl)-1,4a-dihydro-4H-benzofuro[3,2-f]indazol-4-one
(E4)
1
4
4
4
4
4
4
4
56
57
58
59
60
61
62
Yellow crystal. M.p. 183-184℃. Yield: 46%. H-NMR (CDCl , 300 MHz, ppm): δ 13.32 (s, 1H,
3
Ar-OH), 11.14 (s, 1H, Ar-OH), 7.44 (d, J = 8.4 Hz, 2H, Ar-H), 7.34 (d, J = 8.1 Hz, 2H, Ar-H), 6.21
(s, 1H, O=C-CH=), 2.63 (s, 3H, CH -C=O), 2.58 (s, 3H, -N=C-CH ), 2.45 (s, 3H, Ar-CH ), 2.11 (s,
3
3
3
1
3
3H, Ar-CH ), 1.81 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.37, 196.12,
3
3
3
172.72, 163.49, 157.63, 156.27, 151.25, 148.10, 138.93, 135.37, 130.16 (2C), 123.76 (2C), 110.52,
108.17, 104.00, 101.46, 89.35, 60.24, 31.19, 30.43, 21.16, 13.27, 7.45. ESI-HRMS (m/z): calcd for
+
+
C H N O [M+H] : 431.16015; found: 431.16064.
2
5
23
2
5
4
63
64
4
2.8. Procedure for the preparation of compound F1-F4
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The (+)-usnic acid (172 mg, 0.5 mmol), CH COOH (0.125 mL), and different aminoquinolines
3
(0.6 mmol) was stirred in 10 mL absolute ethanol at 78℃ for 12 h. The crude product was obtained
under reduced pressure by evaporation of the solvent after confirming completion of the reaction by
TLC. The silica gel column chromatography was used for products purification (silica gel,
methanol/dichloromethane, 1:250-1:100 v/v). The characteristics of the spectroscopic and physical
data were as shown below.
4
71
4
4
4
72
73
74
2.8.1.
(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-(quinolin-3-ylamino)ethylidene)dibenzo[b,d]furan-1,3
(2H,9bH)-dione (F1)
1
4
4
4
4
4
4
4
4
75
76
77
78
79
80
81
82
Yellow crystal. M.p. 257-258℃. Yield: 62%. H-NMR (CDCl , 300 MHz, ppm): δ 15.42 (brs, 1H,
3
Ar-OH), 13.37 (s, 1H, -C-NH-CH -), 11.63 (brs, 1H, Ar-OH), 8.80 (s, 1H, quinoline-H), 8.18 (d, J
2
= 9.0 Hz, 1H, quinoline-H), 8.02-8.02 (m, 1H, quinoline-H), 7.89-7.80 (m, 2H, quinoline-H),
7.70-7.64 (m, 1H, quinoline-H), 5.94 (s, 1H, O=C-CH=), 2.71 (s, 3H, CH -C=O), 2.67 (s, 3H,
3
1
3
-NH-C-CH ), 2.12 (s, 3H, Ar-CH ), 1.79 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ
3
3
3
3
200.45, 198.96, 191.09, 175.05, 174.15, 163.62, 158.01, 155.64, 147.44, 147.08, 131.47, 130.45,
129.77, 129.55, 128.05, 127.68, 108.36, 104.69, 103.27, 102.12, 101.39, 90.35, 57.70, 31.78, 31.13,
+
+
20.52, 7.41. ESI-HRMS (m/z): calcd for C H N O [M+H] : 471.15506; found: 471.15523.
2
7
23
2
6
4
83
4
4
4
84
85
86
2.8.2.
(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-(quinolin-5-ylamino)ethylidene)dibenzo[b,d]furan-1,3
(2H,9bH)-dione (F2)
1
4
4
4
87
88
89
Yellow crystal. M.p. 120-121℃. Yield: 21%. H-NMR (CDCl , 300 MHz, ppm): δ 15.41 (brs, 1H,
3
Ar-OH), 13.37 (s, 1H, -C-NH-CH -), 11.71 (brs, 1H, Ar-OH), 9.04 (m, 1H, quinoline-H), 8.22 (m,
2
2H, quinoline-H), 7.83-7.78 (m, 1H, quinoline-H), 7.56-7.52 (m, 1H, quinoline-H), 7.46-7.43 (m,
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4
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90
91
92
93
94
1H, quinoline-H), 5.96 (s, 1H, O=C-CH=), 2.72 (s, 3H, CH -C=O), 2.53 (s, 3H, -NH-C-CH ), 2.12
3 3
1
3
(s, 3H, Ar-CH ), 1.82 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ 200.28, 198.83,
3
3
3
191.33, 175.29, 163.57, 158.06, 151.44, 148.64, 130.64, 130.39, 128.78 (2C), 124.50, 124.38,
122.41, 108.29, 105.17, 104.73, 103.01, 102.18, 101.36, 98.27, 57.65, 31.92, 31.29, 20.73, 7.48.
+
+
ESI-HRMS (m/z): calcd for C H N O [M+H] : 471.15506; found: 471.15536.
2
7
23
2
6
4
95
4
4
4
96
97
98
2.8.3.
(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-(quinolin-6-ylamino)ethylidene)dibenzo[b,d]furan-1,3
(2H,9bH)-dione (F3)
1
4
5
5
5
5
5
5
5
99
00
01
02
03
04
05
06
Yellow crystal. M.p. 124-125℃. Yield: 54%. H-NMR (CDCl , 300 MHz, ppm): δ 15.35 (brs, 1H,
3
Ar-OH), 13.37 (s, 1H, -C-NH-CH -), 11.75 (brs, 1H, Ar-OH), 9.02-9.00 (m, 1H, quinoline-H), 8.21
2
(t, J = 9.0 Hz, 2H, quinoline-H), 7.68-7.67 (m, 1H, quinoline-H), 7.57-7.49 (m, 2H, quinoline-H),
5.92 (s, 1H, O=C-CH=), 2.71 (s, 3H, CH -C=O), 2.66 (s, 3H, -NH-C-CH ), 2.12 (s, 3H, Ar-CH ),
3
3
3
1.79 (s, 3H, O=C-C-CH3). 13C-NMR (CDCl , 75 MHz, ppm): δ 200.50, 200.48, 198.87, 191.05,
3
174.88, 173.81, 163.58, 158.07, 155.69, 151.40, 147.13, 135.80, 131.40, 128.24, 127.15, 123.89,
122.26, 108.25, 104.77, 102.94, 102.15, 101.38, 57.64, 31.81, 31.14, 20.66, 7.41. ESI-HRMS (m/z):
+
+
calcd for C H N O [M+H] : 471.15506; found: 471.15576.
2
7
23
2
6
5
07
5
5
5
08
09
10
2.8.4.
(E)-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-(quinolin-8-ylamino)ethylidene)dibenzo[b,d]furan-1,3
(2H,9bH)-dione (F4)
1
5
5
5
5
11
12
13
14
Yellow crystal. M.p. 118-119℃. Yield: 59%. H-NMR (CDCl , 300 MHz, ppm): δ 15.69 (brs, 1H,
3
Ar-OH), 13.38 (s, 1H, -C-NH-CH -), 11.95 (brs, 1H, Ar-OH), 9.04-9.02 (m, 1H, quinoline-H),
2
8.27-8.24 (m, 1H, quinoline-H), 7.88-7.85 (m, 1H, quinoline-H), 7.63-7.61 (m, 2H, quinoline-H),
7.56-7.52 (m, 1H, quinoline-H), 5.95 (s, 1H, O=C-CH=), 2.71 (s, 3H, CH -C=O), 2.68 (s, 3H,
3
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3
5
5
5
5
5
15
16
17
18
19
-NH-C-CH ), 2.12 (s, 3H, Ar-CH ), 1.80 (s, 3H, O=C-C-CH ). C-NMR (CDCl , 75 MHz, ppm): δ
3 3 3 3
200.53, 198.75, 190.91, 173.86, 163.51, 158.23, 151.02, 147.25, 142.69, 136.19, 133.68, 129.01,
127.78, 125.83, 125.24, 122.29, 121.20, 115.90, 109.95, 108.06, 105.06, 102.47, 101.36, 31.72,
+
+
31.16, 21.41, 7.42. ESI-HRMS (m/z): calcd for C H N O [M+H] : 471.15506; found:
2
7
23
2
6
471.15561.
5
5
20
21
2.9. In vitro anti-T. gondii activity
5
5
5
5
5
5
5
5
5
5
5
22
23
24
25
26
27
28
29
30
31
32
The published methyl thiazolyl tetrazole (MTT) method was used to study in vitro the
cytotoxicity of compounds to host cells (HeLa) and the median inhibitory concentration of
compounds to T. gondii-infected HeLa cells. Then the selectivity index (SI) was calculated. Cells
were inoculated with appropriate densities in 96-well plates to ensure that the cells grew
exponentially during the whole experiment period (3,000 cells/well). And then adhered for 24 h at
37℃. Infection of HeLa cells with T. gondii (15,000 tachyzoites/well), and then incubated T.
gondii-infected HeLa cells for 24 h. Dissolved 10 mM compound in DMSO and stored it, and tested
its serial dilution (1-1000 μM). Positive controls used spiramycin (spi), pyrimethamine and
sulfadiazine. After plates were incubated for 24 hours, each well added 10 μL of MTT solution.
Then a further 4 h of incubation. The optical density (OD) was read at 540 nm wavelength from
microplate reader.
5
5
33
34
.
2.10. In vivo anti-T. gondii activity
535
536
537
538
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Divide 36 female KM mice into 6 groups (normal group, infected untreated group, infected
with spi-treated group, infected with (+)-usnic acid-treated group, infected with D3-treated group,
infected with F3-treated group), each group had six mice, and invasion of 2,000 tachyzoites of the
T.gondii RH strain into mice by intraperitoneal injection in vivo. An animal model of acute
infection of T.gondii was prepared by intraperitoneal injection of tachyzoites for 4 hours in mice.
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Then, mice were gavaged once a day for 4 consecutive days with 100 mg/kg of compound, and the
same dose of distilled water was given to the untreated group. After the fifth day of infection in
mice, the mice were sacrificed by cervical dislocation and the abdominal parasites were collected
by rinsing with sterile physiological saline. The inhibition rate of compounds against T.gondii was
calculated by counting the number of tachyzoites in mouse peritoneal fluid using optical
microscopy. Anatomize the liver and spleen. The liver and spleen index, aspartate aminotransferase
(AST), serum alanine aminotransferase (ALT) and malonaldehyde (MDA) and liver homogenate
glutathione (GSH) were measured. And, pathological examination was performed through liver
sections treated with hematoxylin and eosin (H&E).
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2.11. Statistical analysis
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All data were expressed as mean ± standard deviation (SD) triplicate. GraphPad Prism 5.0
(GraphPad Software Inc., San Diego, USA) and SPSS 16.0 software (SPSS Inc., Chicago, USA)
were used for statistical analysis and charts in the study. When p < 0.05, it was considered
statistically signifificant.
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3. Results and discussion
3.1. Chemistry
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(+)-usnic acid has an insecticidal effect in vitro, and trace amounts of (+)-usnic acid exhibit a
strong anti-T. gondii tachyzoite activity [48]. According to the antibacterial and insecticidal
experiments and clinical application results of (+)-usnic acid, it is speculated that (+)-usnic acid
may also have a good anti-insect effect on T. gondii in humans and animals. Conveniently,
(+)-usnic acid was also commercially available, so the (+) isomer was selected in our study.
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The reaction of various primary amines with (+)-usnic acid highly reactive methyl ketone [49]
to form enamine compounds is our strategic basis. Scheme 1. shown the synthetic method for
obtaining target products. Compounds A1-A8 are obtained by the reaction between various
aminotriazoles and (+)-usnic acid in anhydrous ethanol at 78℃, and the yields of A1-A8 ranged
between 47%-66%. Compounds B1-B5 are obtained by the reaction between various 2-aminoethyl
secondary amines and (+)-usnic acid in anhydrous ethanol at 78℃, and the yields of B1-B5 ranged
between 72%-96%. Compounds C1-C5 are obtained by the reaction between various
phosphoramidates and (+)-usnic acid in anhydrous ethanol at 78℃, and the yields of C1-C5 ranged
between 50%-66%. Compounds D1-D4 were obtained by an amide condensation reaction with
different amino acid esters, catalyzed by Et N in ethanol and water at 78℃, and the yields of D1-D4
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ranged between 48%-57%. Compounds E1-E4 are obtained by the reaction between various
benzoquinones and (+)-usnic acid, catalyzed by Et N in anhydrous ethanol at 78℃, and the yields
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of E1-E4 ranged between 44%-70%. Compounds F1-F4 are obtained by the reaction between
different aminoquinolines and (+)-usnic acid, catalyzed by CH COOH in anhydrous ethanol at 78℃,
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and the yields of F1-F4 ranged between 21%-62%.
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Preparation of the intermediates is shown in Scheme 2: The aniline is subjected to
diazotization to obtain a phenyl azide compound, which is then reacted with propynylamine under
.
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the catalysis of CuSO 5H O and sodium ascorbate to obtain intermediates 1a-1h. After reacting a
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mixture of benzaldehyde, diethyl phosphite and amine acetate overnight, HCl was adjusted to pH =
1-4, and NaOH was adjusted to pH = 10, and extracted with dichloromethane to obtain
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intermediates 2a-2e. Characterization of all target compounds by H-NMR spectroscopy before
biological evaluation.
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3.2 Evaluation of anti-T. gondii activity in vitro and andstructure-activity relationship (SAR)
studies
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The IC in T. gondii-infected HeLa cells and IC in HeLa cells were determined by the MTT
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assay and the selectivity index was calculated (Table 1). In vitro, the selectivity index was
calculated using formula: the ratio of the CC value for host cells not infected with T. gondii to the
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IC for T. gondii cultivated in host cells (SI= CC / IC ) [50]. It is generally believed that the
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larger the SI, the stronger the activity against T. gondii and the lower the cytotoxicity [47]. To
provide references for the subsequent in vivo experiments. In this paper, the lead compound used
(+)-usnic acid, positive control drugs used spi, pyrimethamine and sulfadiazine. Can be seen from
Table 1., the cytotoxicity of (+)-usnic acid was lower than spi, and the selectivity index was also
higher which indicated a strong anti-T. gondii potential. Compound A series introduced different
substituted aminotriazoles into the lead (+)-usnic acid, and the sequence of selectivity index was as
follows: p-Br> p-CF > p-F> o-Cl> o-OCH > p-OCH > p-H> p-Cl. To some extent, it suggests that
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for the anti-T. gondii activity,it may be beneficial to introducing strong electron withdrawing groups
in the para position of the benzene ringl. Among these compounds, the cytotoxicity of the
compounds A3, A4, A5, A6 and A8 were lower than (+)-usnic acid, and the selectivity index was
higher, which indicated more anti-T. gondii activity. The compound B series introduced different
substituted 2-aminoethyl secondary amines into the lead (+)-usnic acid. These compounds showed a
significant increased cytotoxicity compared to the lead (+)-usnic acid, and only the compound B2
which the R group was piperidine substituted had the higher selectivity index than that of (+)-usnic
acid. The preliminary structure-activity relationship showd that the nitrogen atoms increasing
reduced the anti-T. gondii activity, while increasing in carbon atoms seemed to increase the activity.
Compound C series introduced different substituted phosphoramidates in the lead (+)-usnic acid.
In this series, C1 showd the strongest cytotoxicity, which indicated that the introduction of
substituents on the benzene ring was beneficial for cytotoxicity reduction. The D series linked four
different amino acid methyl esters on the (+)-usnic acid molecule. Among them, the compound D3
had the highest selectivity index under the premise of low cytotoxicity, The selective index value
was greater than 2.77, anti-T. gondii activity was significantly better than the three positive control
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drugs or the lead, which indicated its value in further research. To some extent, the R group was the
most active when it was a fat-soluble benzyl group, and the activity of the R group which contained
a secondary amine or a phenolic hydroxyl group was weakened. The compound E series linked
different substituted benzoquinones on the (+)-usnic acid. The preliminary structure-activity
relationship indicated that for anti-T. gondii activity, the introduction of an electron withdrawing
group at the meta position of the benzene ring may enhance it. Compound F series linked different
substituted aminoquinolines on the (+)-usnic acid. Among them, compound F3 had relatively low
cytotoxicity and high selectivity index, which indicated its activity was better than the lead or the
three positive control drugs.
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3.3 Number of tachyzoites in vivo
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Assessment of the amount of tachyzoites in the mice peritoneal cavity to verify whether
(+)-usnic acid derivatives D3 and F3 show the effect against T. gondii in vivo. Can be seen from
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Table 2 and Figure 2, untreated KM mice had 2.25 × 10 tachyzoites in the peritoneum. After 100
mg/kg of spi, (+)-usnic acid, D3 and F3 treatment, the tachyzoite inhibition rate in the peritoneal
cavity of mice reached to 55.2% (p < 0.01), 58.3% (p < 0.01), 76.0% (p < 0.001) and 64.6% (p <
0.001), and the tachyzoites number was significantly reduced. At the same concentration, (+) -
usnic acid, D3 and F3 inhibited tachyzoites much more effectively than the positive control spi.
Moreover, compounds D3 and F3 had better anti-T. gondii activity than the natural product
(+)-usnic acid. These results showed that compounds D3 and F3 are effective in inhibiting T. gondii
in vivo, especially D3.
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3.4 Liver and spleen indices
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Liver has been shown to be the main part of tissue pathology during acute, lethal
toxoplasmosis in mice [51], and it is the spleen that plays an irreplaceable role in coordinating
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innate immune responses and adaptive [52]. Therefore, liver and spleen indices were used to assess
the protective action of drugs on internal organs. Calculate the liver and spleen indices by following
formula: viscera index = viscera weight/body weight. Can be seen from Figure 3, There were no
significant differences in the data between each group in the liver index experiment, and it was not
statistically significant. The spleen index of the normal group was significantly lower than the T.
gondii infection group, it was shown that splenomegaly of organs related to the immune system was
caused by acute T. gondii infection (p < 0.05). After administration in mice infected with T. gondii,
splenomegaly was reduced than the normal group, especially in the (+)-usnic acid-treated group (p
< 0.05) and the D3-treated group (p < 0.01). These indicate that splenomegaly caused by T. gondii
infection can be effectively alleviated by compound D3.
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3.5 ALT and AST
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The largest gland organ in the human body is the liver. In addition to detoxifying various
metabolites and synthesizing proteins and biochemicals necessary for digestion, it also has a central
role in the pathophysiology of parasitic infection [53]. The level of serum ALT and AST activity
are very sensitive indicators to evaluate liver injury, and their elevation roughly reflects the degree
of the damage. ALT and AST levels in KM mice serum were measured to further investigate the
toxicity of these compounds (Figure 4). Compared with the normal group, mice serum ALT levels
in the the T. gondii infection group were significantly higher (p < 0.05). Serum ALT levels were
significantly lower (p < 0.01) in the D3 or F3 treatment group and were superior to the (+)-usnic
acid treatment group. Simultaneously, compared with the normal group, serum AST levels in mice
infected with T. gondii was significantly higher (p < 0.001). Serum AST levels were significantly
lower in the D3 treatment group (p < 0.01), and superior to the spi treatment group (p < 0.01).
These results indicate that compound D3 can reduce hepatotoxicity while resisting T. gondii.
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3.6 MDA and GSH
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GSH is a special substance for detoxification. It is an important free radical scavenger and
antioxidant for the person, combining with heavy metals and free radicals to convert harmful toxins
in the body into harmless substances and excrete them out of the body [54]. In vivo, free radicals act
on lipids to enact a peroxidation reaction producing MDA, which can cause the cross-linking
polymerization of other living macromolecules, nucleic acids and proteins, and is cytotoxic. Can be
seen from Figure 5, the GSH content of the T. gondii infection group was significantly lower than
the normal group(p < 0.05). Compared with the T. gondii infection group, the GSH content of the
compounds F3 and D3 treatment group was significantly increased (p < 0.001), and was also better
than the normal group (p < 0.05), and the (+)-usnic acid treatment group (p < 0.01, p < 0.001). T.
gondii infection caused a significant increase in MDA levels (p < 0.01). Compared with the T.
gondii infection group, compound D3 could significantly decrease the MDA content (p < 0.01),
which was the same as spi treatment. These results indicate that compound D3 exerts a strong
antioxidant effect against T. gondii infection.
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3.7 Histological Analysis
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To determine the effect of the compounds on liver, we observed the general changes of the
liver. The color of the livers from the normal group were dark red, while the livers from infected
mouse displayed a white surface. The livers from the groups under treatment showed different
degrees of whitening, and (+)-usnic acid group showed the least significant effect. It indicated that
the (+)-usnic might have potentials on liver protection during acute infection (data not shown).
Furthermore, pathological analysis of sections of livers of mice infected with T. gondii to compare
the anti-T. gondii effect of (+)-usnic acid and its derivatives with the spi-treated positive group
(Figure 6., Table 3.). Staining tissue samples from mice infected with T. gondii with H&E. Liver
specimens from the negative control group showed a moderate level of granuloma; the amount of
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