New Pyrano-4H-benzo[g]chromene-5,10-diones with Antiparasitic and Antioxidant Activities

Article abstract of DOI:10.1002/cbdv.202000839

New pyranonaphthoquinone derivatives were synthesized and investigated for their activity against Trypanosoma brucei, Leishmania major, and Toxoplasma gondii parasites. The pentafluorophenyl derivative was efficacious against T. brucei with single digit m

Full text of DOI:10.1002/cbdv.202000839

Title: New pyrano-4H-benzo[g]chromene-5,10-diones with anti-
parasitic and anti-oxidant activities
Authors: Ibrahim S. Al Nasr, Jana Jentzsch, Amin Shaikh, Priti Singh
Shuveksh, Waleed S. Koko, Tariq A. Khan, Khursheed
Ahmed, Rainer Schobert, Klaus Ersfeld, and Bernhard
Biersack
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000839
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10.1002/cbdv.202000839
Chemistry & Biodiversity
Chem. Biodiversity
New pyrano-4H-benzo[g]chromene-5,10-diones with anti-parasitic and anti-
oxidant activities
Ibrahim S. Al Nasr,a,b Jana Jentzsch,c Amin Shaikh,d Priti Singh Shuveksh,d Waleed S. Koko,b Tariq A. Khan,e
Khursheed Ahmed,d Rainer Schobert,f Klaus Ersfeld,c and Bernhard Biersack *,f
a Department of Biology, College of Science and Arts, Qassim University, Unaizah 51911, Saudi Arabia
b Department of Science Laboratories, College of Science and Arts, Qassim University, Ar Rass 51921, Saudi Arabia
c Laboratory of Molecular Parasitology, University of Bayreuth, Universitätsstrasse 30, 95440 Bayreuth, Germany
d Department of Chemistry, Abeda Inamdar Senior College, 2390-B, K.B. Hidayatullah Road, Pune 411001, India
e Department of Clinical Nutrition, College of Health Sciences, Qassim University, Ar Rass 51921, Saudi Arabia
f Organic Chemistry 1, University of Bayreuth, Universitätsstrasse 30, 95440 Bayreuth, Germany, e-mail: bernhard.biersack@yahoo.com
New pyranonaphthoquinone derivatives were synthesized and investigated for their activity against Trypanosoma brucei, Leishmania major, and
Toxoplasma gondii parasites. The pentafluorophenyl derivative was efficacious against T. brucei with single-digit micromolar EC50 values and against T.
gondii with even sub-micromolar values. The 3-chloro-4,5-dimethoxyphenyl derivative showed an activity against amastigotes of Leishmania major
parasites comparable to that of amphotericin B. In addition, antioxidant activities were observed for the bromophenyl derivatives, and their redox behavior
was studied by cyclovoltammetry. Anti-parasitic and antioxidative activities of the new naphthoquinone derivatives appear uncorrelated.
Keywords: lawsone • neglected tropical diseases • pyran • biological activity
Introduction
Infections of immune-compromised patients and new-born children with the world-wide occurring toxoplasmosis (caused by Toxoplasma gondii parasites)
can lead to severe complications and, thus, efficient drugs for the treatment of toxoplasmosis are necessary.[1] In addition, neglected tropical diseases
(NTDs) pose an eminent danger to people living or working in affected territories.[2,3] Human African trypanosomiasis (HAT, sleeping sickness) and
leishmaniasis are NTDs which can ultimately lead to death of untreated patients. Trypanosoma brucei gambiense (T. b. gambiense, in West and Central
Africa) and T. b. rhodesiense (in East Africa) are the two prevalent forms of trypanosomes which are responsible for sleeping sickness in humans while the
Nagana disease of cattle (animal trypanosomiasis) is mainly caused by T. b. brucei.[4,5] The diamidine pentamidine and the urea derivative suramin are drugs
only applicable for patients with early stage sleeping sickness, late stages were treated with highly toxic arsenics such as melarsoprol for a long time.[6]
Meanwhile, the less toxic ornithine derivative eflornithine in combination with the nitrofuran nifurtimox (nifurtimox-eflornithine combination treatment,
NECT) has replaced melarsoprol for the treatment of late stage T. b. gambiense infections (g-HAT).[6] The nitroimidazole fexinidazole was the first orally
active drug which is applied for the treatment of stage 1 and stage 2 g-HAT.[5] The benzoxaborole derivative acoziborole is currently tested in phase 2/3
clinical trials with stage 2 g-HAT patients.[5] Leishmaniasis is clinically subdivided into the cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL)
and visceral leishmaniasis (VL) forms, which show different clinical outcomes. VL is caused bei Leishmania infantum (L. infantum) and L. donovani protozoal
parasites and eventually leads to death resulting into a severe medical problem in regions where VL is endemic, e.g., in South Asia, Africa, Latin America
and the Mediterranean region.[7] The liposomal formulation of the natural polyene macrolide amphotericin B (AmBisome) and combinations of AmBisome
with the phospholipid miltefosine are currently applied for the treatment of VL.[7] The skin damaging CL form, which is caused by various Leishmania species
such as L. major, L. tropica, L. mexicana, L. amazonensis etc., is the most widespread form of leishmaniasis diseases with 0.7-1 million predominantly young
patients every year.[8,9] Although CL is usually not lethal, it causes severe and disfiguring skin lesions and affected persons are often stigmatized.[10,11] Current
treatment options for CL patients include pentavalent antimonials (sodium stibogluconate, meglumine antimoniate), miltefosine, amphotericin, and
pentamidine. Recent works disclosed interesting preclinical results for benzoxaborole, nitroimidazoles and aminopyrazoles as well as for the antimonial
drug activity enhancer D35 (a CpG oligonucleotide).[9,12] Aside of the toxicity of the currently applied drugs, the emergence of drug-resistant parasite forms
poses a growing problem to the clinician and, thus, the search for new potent anti-parasitic drugs is ongoing. The treatment of trypanosomiasis and
leishmaniasis with natural products or with drugs derived from them appears promising.[3,13] Naphthoquinones represent a significant group of secondary
metabolites of plants and lichens with a variety of biological activities such as antioxidant and trypanocidal activities.[3,13-15] The natural 2-hydroxy-1,4-
naphthoquinone (lawsone, 1a) isolated from the Henna plant Lawsonia inermis showed biological activities including antibacterial effects and was applied
1
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10.1002/cbdv.202000839
Chemistry & Biodiversity
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as starting material for quinone drug candidates such as lapachol and atovaquone (ATO).[3,13-16] Modified lawsone derivatives with antitumor and antifungal
activities are also known.[3,17,18] Quite a few lawsone derivatives were found efficacious against various parasites including Plasmodium falciparum,
Leishmania donovani, Trypanosoma cruzi and Toxoplasma gondii.[3,19-23] Our own previous works have led to the identification of lawsone Mannich bases and
bis-naphthoquinone derivatives (1b–d) with reasonable activities against Trypanosoma brucei, Entamoeba histolytica, L. major or T. gondii (Figure 1).[3,24,25]
The fusion of the lawsone scaffold with heterocyclic rings such as pyrans can be another promising method in order to obtain new active compounds based
on lawsone and various anti-tumour active derivatives as well as tumour cell senescence-inducing complexes were already identified (1e).[26,27] In
continuation of these previous reports, we present new lawsone-derived pyrano-naphthoquinone derivatives and their antioxidant and anti-parasitic
activities against T. brucei, L. major and T. gondii.
Figure 1. Lawsone 1a, selected anti-parasitic derivatives 1b–d, and anti-tumour active pyran 1e.
Results and Discussion
Compounds 2a–o were prepared by Knoevenagel reaction of malononitrile and the respective aryl aldehyde under basic conditions followed by Michael
addition of lawsone (1a) and ring-closure to the pyran ring in a multi-component one-pot reaction (Scheme 1).[25,26] Compounds 2a–o were obtained as
yellow, brown or red-orange solids in moderate yields.
Scheme 1. Reagents and conditions: (a) Et3N, r.t., 30 min – 16 h, yields in brackets.
The compounds 2a–o were tested against T. gondii parasites and their activities were compared with those against non-malignant Vero cells (Table 1).
Except for 2a and 2c, all test compounds exhibited distinct activity against T. gondii. Compound 2h showed the highest activity in the sub-micromolar range
(EC50 = 0.7 µM) and a reasonable selectivity (SI = 19.1). Compounds 2g and 2o showed similar selectivities (SI = 19.6 for 2g, 22.1 for 2o). Considerably higher
selectivities were observed for 2d (SI = 34.5), 2f (SI = 34.9), and 2l (SI = 41.4). Although the selectivity of these compounds is lower when compared with the
selectivity of the approved drug ATO, they were less toxic than ATO against Vero cells and, thus, they can be less toxic alternatives to ATO treatment.
2
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Table 1. Antitoxoplasmal activity of compounds 2a-o as EC50 (effective concentration that causes 50% inhibition for T. gondi in µM)[ a], while IC50 is the inhibition concentration
for 50% of the Vero (African green monkey kidney epithelial in µM). ATO was used as positive control.
Compd.
EC50 (T. gondii)
IC50 (Vero)
SI (Vero / T. gondii)[b]
2a
131 ± 13
4.1 ± 0.6
0.03
0.43
0.57
34.5
6.92
34.9
19.6
19.1
6.26
9.94
9.92
41.4
6.44
12.64
22.1
136
2b
1.7 ± 0.2
10.6 ± 1.7
1.7 ± 0.18
2.5 ± 0.3
2.2 ± 0.25
1.8 ± 0.22
0.7 ± 0.1
2.8 ± 0.32
4.5 ± 0.46
3.1 ± 0.38
1.3 ± 0.5
1.4 ± 0.42
1.4 ± 0.53
2.8 ± 0.35
0.07 ± 0.004
-
0.7 ± 0.08
6.0 ± 0.72
59.8 ± 7.4
17.1 ± 2.3
76.6 ± 8.7
35.8 ± 4.4
13.7 ± 1.5
17.4 ± 2.4
45.1 ± 6.2
30.3 ± 3.9
54.6 ± 6.2
9.1 ± 1.4
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
17.9 ± 2.1
62.7 ± 6.0
9.5 ± 1.54
0.4 ± 0.01[c]
2o
ATO
Doxorubicin
-
[a] Values are the average of three repeated reading for each test ± SE. Which were obtained from concentration–response curves by measuring the percentage of vital
cells relative to untreated group after 3 days of incubation. [b] Selectivity index (SI) calculated by dividing IC50 over EC50 of the corresponding values. [c] Value is taken from
ref. 28.
Next, compounds 2a–o were investigated against L. major promastigotes and amastigotes and their activities were compared with those against non-
malignant Vero cells (Table 2). Compound 2b showed excellent activity against L. major amastigotes (EC50 = < 0.5 µM) and, thus, 2b was at least comparably
active when compared with the approved drug amphotericin B (AmB) in this regard. However, 2b was also quite toxic to Vero cells (i.e., it was almost as
toxic as doxorubicin) leading to a relatively low selectivity when compared with AmB.[28] In addition, compounds 2a, 2e, 2h, 2i, and 2m showed activities
against the amastigotes with EC50 values below 10 µM. Compounds 2d (SI = 4.22) and 2o (SI = 5.26) revealed the highest selectivities for L. major amastigotes.
Compound 2h also showed moderate activity against L. major promastigotes (EC50 = 15.3 µM). However, the activity of the test compounds 2a–o against
promastigotes was distinctly lower when compared with their activities against amastigotes.
Table 2. Antileishmanial activity of compounds 2a-o as EC50 (effective concentration that causes 50% inhibition for L. major amastigotes and promastigotes in µM)[a], AmB was
used as positive control.
Compound
EC50 promastigotes
EC50 amastigotes
SI Vero / promastigotesb
SI Vero / amastigotes[b]
2a
2b
2c
49.2 ± 5.2
34.1 ± 4.4
22.3 ± 3.1
146 ± 16.5
37.9 ± 4.7
86.5 ± 9.1
25.6 ± 3.6
15.3 ± 1.9
35.8 ± 4.0
98.0 ± 10.1
46.4 ± 5.2
84.1 ± 9.3
29.4 ± 3.6
37.9 ± 4.5
41.2 ± 5.0
0.83 ± 0.09
8.4 ± 1.1
< 0.5
0.08
0.02
0.27
0.41
0.45
0.89
1.40
0.89
0.49
0.46
0.65
0.65
0.31
0.47
1.52
9.6
0.49
>1.5
0.17
4.22
2.31
2.45
3.19
2.38
1.77
1.32
1.24
2.46
1.15
0.67
5.26
16.4
35.8 ± 4.3
14.2 ± 1.6
7.4 ± 0.9
31.3 ± 3.4
11.3 ± 0.9
5.7 ± 0.8
9.8 ± 1.1
34.2 ± 4.7
24.5 ± 3.9
22.2 ± 2.8
7.9 ± 1.0
26.6 ± 2.6
11.9 ± 2.1
0.47 ± 0.06
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
AmB
[a] Values are the average of three repeated reading for each test ± SE. Which were obtained from concentration–response curves by measuring the percentage of vital cells
relative to untreated group after 3 days of incubation. [b] Selectivity index (SI) calculated by dividing IC50 (from Table 1) over EC50 of the corresponding values.
3
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Selected compounds were also tested against T. b. brucei (Table 3). Pentamidine served as positive control here.[29] Compound 2h showed the highest
activities against the T. b. brucei cells followed by 2f and 2a. The highest selectivity was observed for 2f (SI = 12.8). Compounds 2b, 2c, and 2m were inactive
at doses up to 10 µM.
Table 3. Antitrypanosomal activity of compounds 2a–c, 2f, 2h and 2m as EC50 (effective concentration that causes 50% inhibition for T. b. brucei in µM)[a], pentamidine was used
as positive control.
Compd.
IC50 (T. b. brucei)
SI Vero / T. b. brucei[b]
2a
7.6
0.54
2b
> 10
-
2c
> 10
-
2f
6.0
12.8
2h
4.9
2.8
2m
> 10
-
-
Pentamidine
0.000042[c]
[a] Values are the means of at least three independent experiments (SD ± 15%). They were derived from concentration–response curves obtained by measuring the
percentage of vital cells relative to untreated controls after 72 h. [b] Selectivity index calculated from the corresponding IC50 values for the Vero cells and the IC50 values
for T. b. brucei. [c] Value is taken from ref. 29.
The antioxidant activities of selected compounds 2a–f, 2h, 2m and 2n were evaluated using the 1,1-diphenyl-2-picrylhydrazil (DPPH) assay (Table 4). The
radical derivative DPPH is a radical scavenger and functions as a trap for other radicals and, thus, it is the functional compound of common antioxidant
assays.[30-32] Compounds 2a and 2c showed distinctly higher antioxidant activities when compared with the other test compounds. Both compounds were
also more active than the known antioxidant ascorbic acid. There seems to be no or just a marginal correlation between antioxidant activity and anti-
parasitic activity of the test compounds.
Table 4. Inhibitory concentrations IC50 (in µM) of ascorbic acid (positive control) and test compounds 2a–f, 2h, 2m, and 2n when tested for their antioxidant activities (DPPH
assay).
Compdound
IC50
Ascorbic acid
20
2a
2b
2c
3.75
22
2.25
28
2d
2e
2f
16
21
2h
2m
2n
25
20
30
The cell-independent redox properties of lawsone derivatives 2a to 2o were studied by cyclic voltammetry (Figure S1-S7). 2a gave rise to a pair of peaks at
–358 mV (ipa −0.889 µA) and −556 mV (ipc 1.792 µA) indicative of a three-electron transfer redox couple with E1/2 = −457 mV. While 2c and 2h showed similar
redox couples with almost the same ΔE values, these compounds displayed another irreversible cathodic peak between −1004 mV and 1050 mV with
different current values (ipc 0.3104 µA in case of 2c). Such a redox behaviour was reported for other lawsone derivatives and the redox couple at E1/2 = –457
mV can be assigned to the conversion of naphthoquinone (NQ) to naphthosemiquinone (NSQ) (NQ
NSQ), while the irreversible peak at 1050 mV can
be assigned to the following 2e- reduction to the catechol form (NSQ  CAT).[33-35] The shift of this redox couple towards a more negative potential when
compared to the parent lawsone compound can be attributed to the changes occurring in the electron density based on the bromine atoms of 2a and 2c.
The electron distributions in redox active ligands were correlated with a preferential reduction of certain ligands.[36-38] The π-orbital of the
benzosemiquinone-type radical ligand of 2c having a large overlap area might contribute to its greater ROS producing effects and, thus, showed the highest
antioxidant activity compared to the other test compounds. In case of 2d, a large gain in the current (ipc 1.3047 µA and ipa –1.4905 µA) was probably due to
the electron withdrawing effects of the fluorine atom and, thus, a fast electron transfer was facilitated (Figure S1).[36] Compound 2h showed a redox couple
at E1/2 = –428.5 mV with high current values (ipc 1.9638 µA and ipa -1.0944 µA).
4
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Conclusions
New lawsone-derived compounds were identified as anti-parasitic and antioxidant agents. The high anti-parasitic activities of 2h against T. brucei and T.
gondii warrant a further investigation of its effects on these parasites and other protozoal parasites such as Plasmodium species. The highest antioxidant
activities, i.e., the strongest suppression of radical formation, were observed for compounds 2a and 2c, which showed relatively weak activities in the anti-
parasitic assays (except for 2a against T. brucei) and so there is apparently no strong correlation between antioxidant and anti-parasitic activities of the test
compounds.
Experimental Section
General
Starting materials and pure solvents were purchased from common providers and used without further purification. IR spectra were measured on a
PerkinElmer Spectrum One FT-IR spectrophotometer equipped with an ART sampling unit. NMR spectra were measured on a Bruker Avance 300
spectrometer and chemical shifts (δ) are given in parts per million (ppm) downfield from Me4Si as internal standard. Coupling constants (J) are given in Hz.
Mass spectra were measured on a Varian MAT 311A (EI). Elemental analyses were carried out with a Perkin-Elmer 2400 CHN elemental analyser.[3]
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-5,10-dihydro-5,10-dioxo-4H-benzo[g]chromene-3-carbonitrile
(2a).
3-Bromo-4,5-
dimethoxybenzaldehyde (245 mg, 1.0 mmol) and malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The
reaction mixture was stirred at room temperature for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was
stirred at room temperature for 16 h. The formed precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 262 mg (0.56
mmol, 56%); orange solid of m.p. 250-251°C; IR: 3392, 3319, 3256, 3222, 3201, 3017, 2989, 2940, 2825, 2201, 1666, 1655, 1637, 1607, 1591, 1567, 1488, 1460,
1442, 1403, 1363, 1316, 1302, 1278, 1241, 1226, 1206, 1177, 1136, 1077, 1037, 1001, 949, 866, 819, 769, 747, 732, 721, 712, 680; 1H-NMR (300 MHz, DMSO-d6)
δ 3.70 (3 H, s), 3.81 (3 H, s), 4.61 (1 H, s), 7.01 (1 H, s), 7.11 (1 H, s), 7.36 (2 H, s), 7.8-7.9 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, DMSO-d6) δ 36.2, 56.2,
57.1, 60.0, 112.2, 116.7, 119.3, 120.8, 123.1, 125.8, 126.0, 130.8, 131.1, 134.1, 134.4, 141.2, 144.6, 149.3, 153.3, 158.3, 176.8, 182.7; EI-MS: 468 (100) [M+], 466
(97) [M+], 437 (11), 435 (11), 387 (72), 251 (92); Anal. calc. for C22H15BrN2O5 (467.28): C 56.55, H 3.24, N 6.00; found: C 56.41, H 3.16, N 5.88.
2-Amino-4-(3-chloro-4,5-dimethoxyphenyl)-5,10-dihydro-5,10-dioxo-4H-benzo[g]chromene-3-carbonitrile
(2b).
3-Chloro-4,5-
dimethoxybenzaldehyde (200 mg, 1.0 mmol) and malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The
reaction mixture was stirred at room temperature for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was
stirred at room temperature for 16 h. The formed precipitate was collected, washed with H2O/MeCN and dried in vacuum. Yield: 200 mg (0.47 mmol, 47%);
orange solid of m.p. 306-307°C; IR: 3393, 3323, 3256, 3222, 3200, 2991, 2941, 2827, 2202, 1668, 1655, 1638, 1592, 1572, 1492, 1448, 1428, 1417, 1404, 1364,
1341, 1331, 1318, 1303, 1282, 1242, 1228, 1207, 1179, 1161, 1094, 1079, 1046, 1028, 1001, 949, 868, 851, 844, 818, 7973 784, 771, 752, 733, 722, 697, 682, 631,
1
602; H-NMR (300 MHz, DMSO-d6) δ 3.71 (3 H, s), 3.81 (3 H, s), 4.62 (1 H, s), 6.9-7.0 (2 H, m), 7.35 /2 H, s), 7.8-7.9 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5
MHz, DMSO-d6) δ 36.2, 56.2, 57.1, 60.1, 111.5, 119.2, 120.4, 120.7, 125.8, 126.0, 126.8, 130.8, 131.0, 134.1, 134.4, 140.6, 143.6, 149.3, 153.4, 158.3, 176.8,
182.6; EI-MS: 424 (34) [M+], 422 (100) [M+], 387 (63), 251 (77); Anal. calc. for C22H15ClN2O5 (422.82): C 62.50, H 3.58, N 6.63; found: C 62.29, H 3.46, N 6.46.
2-Amino-4-(3,5-dibromo-4-methoxyphenyl)-5,10-dihydro-5,10-dioxo-4H-benzo[g]chromene-3-carbonitrile
(2c).
3,5-Dibromo-4-
methoxybenzaldehyde (293 mg, 1.0 mmol) and malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The
reaction mixture was stirred at room temperature for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was
stirred at room temperature for 16 h. The formed precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 260 mg (0.50
mmol, 50%); orange-red solid of m.p. 267°C; IR: 3414, 3323, 3281, 3253, 3211, 3194, 2201, 1658, 1638, 1606, 1594, 1547, 1471, 1417, 1401, 1362, 1334, 1300,
1
1262, 1203, 1183, 1100, 1074, 1040, 1025, 991, 948, 800, 782, 733, 717, 695, 668; H-NMR (300 MHz, CDCl3/DMSO-d6) δ 3.74 (3 H, s), 4.60 (1 H, s), 6.85 (2 H,
s), 7.40 (2 H, s), 7.7-7.8 (2 H, m), 7.9-8.0 (1 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, CDCl3/DMSO-d6) δ 35.3, 56.8, 59.9, 117.6, 118.4, 121.5, 125.9, 126.0,
129.9, 130.7, 131.7, 133.6, 134.2, 140.9, 148.1, 152.6, 158.6, 176.4, 181.9; EI-MS: 518 (40) [M+], 516 (72) [M+], 514 (40) [M+], 437 (16), 435 (15), 251 (100); Anal.
calc. for C21H12Br2N2O4 (516.15): C 48.87, H 2.34, N 5.43; found: C 48.69, H 2.24, N 5.30.
2-Amino-5,10-dihydro-5,10-dioxo-4-(2-fluorophenyl)-4H-benzo[g]chromene-3-carbonitrile (2d). 2-Fluorobenzaldehyde (124 mg, 1.0 mmol) and
malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The reaction mixture was stirred at room temperature
for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for 90 min. The formed
precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 207 mg (0.60 mmol, 60%); orange solid of m.p. 250-251 °C; IR: 3401,
3318, 3253, 3215, 3190, 2197, 1685, 1664, 1634, 1601, 1579, 1488, 1453, 1407, 1364, 1328, 1303, 1247, 1227, 1207, 1174, 1150, 1098, 1076, 1025, 951, 845, 779,
756, 745, 714, 671; 1H-NMR (300 MHz, DMSO-d6) δ 4.91 (1 H, s), 7.1-7.5 (6 H, m), 7.8-7.9 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, DMSO-d6) δ 30.4, 56.0,
5
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Chemistry & Biodiversity
Chem. Biodiversity
115.3-115.6 (m), 119.1, 120.9, 124.8, 125.8, 126.1, 129.1-129.2 (m), 130.2-130.9 (m), 134.2-134.6 (m), 149.5, 158.0, 159.7 (d, J = 246 Hz), 176.8, 182.5; EI-MS:
346 (100) [M+], 302 (6), 251 (87), 223 (7), 173 (7), 105 (5), 76 (5); Anal. calc. for C20H11FN2O3 (346.32): C 69.36, H 3.20, N 8.09; found: C 69.44, H 3.12, N 7.96.
2-Amino-4-(3,4-difluorophenyl)-5,10-dihydro-5,10-dioxo-4H-benzo[g]chromene-3-carbonitrile (2e). 3,4-Difluorobenzaldehyde (142 mg, 1.0 mmol) and
malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The reaction mixture was stirred at room temperature
for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for 16 h. The formed
precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 151 mg (0.42 mmol, 42%); orange solid of m.p. 260°C; IR: 3403,
3331, 3290, 3256, 3195, 3073, 2206, 1664, 1656, 1639, 1620, 1606, 1594, 1581, 1517, 1438, 1412, 1363, 1332, 1302, 1284, 1274, 1244, 1204, 1179, 1153, 1117,
1
1096, 1076, 1039, 1021, 972, 950, 933, 900, 882, 865, 838, 820, 799, 790, 772, 763, 752, 730, 713, 673; H-NMR (300 MHz, DMSO-d6) δ 4.67 (1 H, s), 7.2-7.5 (5
H, m), 7.8-7.9 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, DMSO-d6) δ 35.8, 56.9, 116.6-117.5 (m), 119.1, 120.7, 124.6, 125.8, 126.0, 130.7, 131.0, 134.1,
134.5, 141.4-141.5 (m), 146.8-147.8 (m), 149.3, 150.0-151.1 (m), 158.2, 176.8, 182.6; EI-MS: 364 (100) [M+], 320 (6), 251 (91), 223 (8); Anal. calc. for C20H10F2N2O3
(364.31): C 65.94, H 2.77, N 7.69; found: C 66.02, H 2.61, N 7.80.
2-Amino-4-(3,5-difluorophenyl)-5,10-dihydro-5,10-dioxo-4H-benzo[g]chromene-3-carbonitrile (2f). 3,5-Difluorobenzaldehyde (142 mg, 1.0 mmol) and
malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The reaction mixture was stirred at room temperature
for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for 16 h. The formed
precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 165 mg (0.45 mmol, 45%); orange-red solid of m.p. 270°C; IR: 3395,
3315, 3249, 3211, 3190, 3090, 2200, 1663, 1622, 1594, 1580, 1463, 1449, 1404, 1363, 1338, 1311, 1303, 1290, 1242, 1206, 1178, 1124, 1077, 1040, 1026, 1005,
1
991, 963, 944, 863, 838, 817, 772, 737, 728, 710, 682, 674; H-NMR (300 MHz, DMSO-d6) δ 4.70 (1 H, s), 7.0-7.2 (3 H, m), 7.41 (2 H, s), 7.8-7.9 (3 H, m), 8.0-8.1
(1 H, m); 13C-NMR (75.5 MHz, DMSO-d6) δ 36.2, 56.6, 102.2-102.9 (m), 110.8-111.1 (m), 119.0, 120.2, 125.8, 126.0, 130.8, 131.0, 134.1, 134.4, 148.1-148.3 (m),
149.6, 158.3, 162.4 (dd, J = 13.1 Hz, 247 Hz), 176.7, 182.6; EI-MS: 364 (96) [M+], 251 (100); Anal. calc. for C20H10F2N2O3 (364.31): C 65.94, H 2.77, N 7.69; found:
C 66.00, H 2.63, N 7.78.
2-Amino-5,10-dihydro-5,10-dioxo-(2,4,5-trifluorophenyl)-4H-benzo[g]chromene-3-carbonitrile (2g). 2,4,5-Trifluorobenzaldehyde (160 mg, 1.0 mmol)
and malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (3 mL) and five drops of Et3N were added. The reaction mixture was stirred at room
temperature for 30 min. Lawsone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for 30 min. The formed
precipitate was collected, washed with MeCN and dried in vacuum. Yield: 120 mg (0.31 mmol, 31%); orange solid of m.p. 274-275°C; IR: 3411, 3318, 3220,
3194, 3059, 2208, 1660, 1640, 1626, 1606, 1593 ,1515, 1427, 1412, 1369, 1332, 1316, 1302, 1266, 1245, 1208, 1185, 1158, 1142, 1094, 1070, 1019, 950, 899, 874,
846, 816, 761, 741, 715, 670, 620; 1H-NMR (300 MHz, CDCl3/DMSO-d6)  4.85 (1 H, s), 6.46 (2 H, s), 6.8-6.9 (1 H, m), 7.0-7.1 (1 H, m), 7.6-7.7 (2 H, m), 7.8-7.9
(1 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, CDCl3/DMSO-d6)  30.7, 56.4, 105.1-105.7 (m), 117.1-117.4 (m), 118.0, 120.5, 125.9, 126.0, 129.9, 130.6, 133.5,
134.1, 148.5, 158.5, 176.3, 181.8; EI-MS: 382 (100) [M+], 251 (92); Anal. calc. for C20H9F3N2O3 (382.30): C 62.84, H 2.37, N 7.33; found: C 62.75, H 2.29, N 7.24.
2-Amino-5,10-dihydro-5,10-dioxo-(2,3,4,5,6-pentafluorophenyl)-4H-benzo[g]chromene-3-carbonitrile (2h). Pentafluorobenzaldehyde (196 mg, 1.0
mmol) and malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (3 mL) and five drops of Et3N were added. The reaction mixture was stirred at room
temperature for 30 min. Lawsone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for 30 min. The formed
precipitate was collected, washed with MeCN and dried in vacuum. Yield: 131 mg (0.31 mmol, 31%); Orange solid of m.p. 281-282°C; IR: 3427, 3286, 3174,
2202, 1669, 1635, 1520, 1505, 1412, 1366, 1330, 1302, 1247, 1207, 1154, 1118, 1079, 1041, 1024, 992, 954, 937, 824, 790, 768, 719, 704, 648, 610; 1H-NMR (300
MHz, DMSO-d6)  5.13 (1 H, s), 7.66 (2 H, s), 7.8-8.0 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, DMSO-d6)  26.8, 52.6, 115.8, 118.7- 118.9 (m), 125.9,
126.2, 129.7, 130.4, 130.9, 135.3, 138.4, 143.0, 146.4, 149.3, 159.5, 176.6, 182.2; EI-MS: 418 (100) [M+], 251 (75); Anal. calc. for C20H7F5N2O3 (418.28): C 57.43,
H 1.69, N 6.70; found: C 57.30, H 1.62, N 6.61.
2-Amino-4-(3,4-dichlorophenyl)-5,10-dihydro-5,10-dioxo-4H-benzo[g]chromene-3-carbonitrile (2i). 3,4-Dichlorobenzaldehyde (175 mg, 1.0 mmol) and
malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The reaction mixture was stirred at room temperature
for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for 16 h. The formed
precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 135 mg (0.34 mmol, 34%); orange-red solid of m.p. 283-285°C; IR:
3396, 3328, 3196, 2203, 1658, 1638, 1605, 1593, 1471, 1411, 1362, 1333, 1294, 1243, 1207, 1177, 1157, 1127, 1093, 1079, 1029, 1017, 973, 948, 872, 834, 822, 793,
772, 750, 738, 728, 715, 682, 659, 617, 583; 1H-NMR (300 MHz, DMSO-d6) δ 4.69 (1 H, s), 7.36 (1 H, dd, J = 8.8 Hz, 2.2 Hz), 7.40 (2 H, s), 7.64 (1 H, dd, J = 8.3 Hz,
2.4 Hz), 7.42 (2 H, s), 7.57 (1 H, d, J = 8.3 Hz), 7.64 (1 H, d, J = 2.4 Hz), 7.8-7.9 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, DMSO-d6) δ 35.8, 56.7, 119.1, 120.4,
125.8, 126.0, 128.3, 129.7, 129.8, 130.6, 130.8, 131.0, 131.1, 134.1, 134.5, 144.8, 149.4, 158.3, 176.8, 182.6; EI-MS: 398 (15) [M+], 396 (23) [M+], 251 (61), 57
(100); Anal. calc. for C20H10Cl2N2O3 (397.21): C 60.48, H 2.54, N 7.05; found: C 60.32, H 2.48, N 7.11.
2-Amino-5,10-dihydro-5,10-dioxo-4-(4-methylthiophenyl)-4H-benzo[g]chromene-3-carbonitrile (2j). 4-Methylsulfanylbenzaldehyde (152 mg, 1.0
mmol) and malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The reaction mixture was stirred at room
temperature for 30 min. The formed precipitate was dissolved by heating the reaction mixture and 2-hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was
added and the reaction mixture was stirred at room temperature for 16 h. The formed precipitate was collected, washed with MeCN and n-hexane and
6
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10.1002/cbdv.202000839
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dried in vacuum. Yield: 280 mg (0.75 mmol, 75%); orange-red solid of m.p. 266°C; IR: 3398, 3319, 3248, 3211, 2194, 1691, 1655, 1638, 1604, 1593, 1494, 1409,
1
1365, 1336, 1303, 1238, 1208, 1179, 1162, 1097, 1077, 1041, 1026, 986, 986, 956, 945, 846, 833, 800, 771, 741, 720, 686; H-NMR (300 MHz, DMSO-d6) δ 2.43
(3 H, s), 4.58 (1 H, s), 7.19 (2 H, d, J = 8.6 Hz), 7.26 (2 H, d, J = 8.6 Hz), 7.33 (2 H, s), 7.8-7.9 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, DMSO-d6) δ 14.6,
36.0, 57.3, 119.3, 121.8, 125.8, 126.1, 128.3, 130.6, 131.0, 134.1, 134.5, 148.8, 158.3, 176.9, 182.6; EI-MS: 374 (100) [M+], 327 (45), 251 (69); Anal. calc. for
C21H14N2O3S (374.41): C 67.37, H 3.77, N 7.48; found: C 67.22, H 3.66, N 7.37.
2-Amino-5,10-dihydro-5,10-dioxo-(3-fluoro-4-methylthiophenyl)-4H-benzo[g]chromene-3-carbonitrile (2k). 3-Fluoro-4-methylsulfanylbenzaldehyde
(170 mg, 1.0 mmol) and malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The reaction mixture was
stirred at room temperature for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room
temperature for 16 h. The formed precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 240 mg (0.61 mmol, 61%);
orange-red solid of m.p. 244-245°C; IR: 3402, 3320, 3251, 3212, 3071, 3046, 2937, 2896, 2195, 1691, 1663, 1665, 1639, 1603, 1593, 1563, 1481, 1412, 1363,
1336, 1300, 1279, 1238, 1207, 1179, 1153, 1096, 1065, 1040, 1026, 954, 931, 881, 838, 822, 803, 776, 761, 736, 719, 695, 685, 619, 561, 553; 1H-NMR (300 MHz,
DMSO-d6) δ 2.45 (3 H, s), 4.64 (1 H, s), 7.1-7.3 (3 H, m), 7.36 (2 H, s), 7.8-7.9 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, DMSO-d6) δ 14.0, 35.8, 57.0, 114.2-
114.5 (m), 119.1, 120.9, 123.7, 124.0, 124.4, 125.8, 126.0, 127.5, 130.7, 131.0, 134.1, 134.4, 142.9, 149.2, 158.3, 158.9 (d, J = 242 Hz), 176.8, 182.6; EI-MS: 392
(100) [M+], 345 (28), 251 (82); Anal. calc. for C21H13FN2O3S (392.40): C 64.28, H 3.34, N 7.14; found: C 64.09, H 3.26, N 7.05.
2-Amino-5,10-dihydro-5,10-dioxo-4-(3-pentafluorothiophenyl)-4H-benzo[g]chromene-3-carbonitrile (2l). 3-Pentafluorothiobenzaldehyde (232 mg,
1.0 mmol) and malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (5 mL) and three drops of Et3N were added. The reaction mixture was stirred at
room temperature for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for
16 h. The formed precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 185 mg (0.41 mmol, 41%); orange-red solid of
m.p. 283°C; IR: 3413, 3340, 3256, 3222, 3194, 3076, 2201, 1656, 1637, 1591, 1484, 1435, 1412, 1361, 1336, 1300, 1252, 1243, 1203, 1185, 1161, 1112, 1095, 1072,
1
1022, 945, 881, 839, 823, 805, 793, 780, 731, 718, 699, 687, 678; H-NMR (300 MHz, DMSO-d6) δ 4.84 (1 H, s), 7.43 (2 H, s), 7.5-7.6 (1 H, m), 7.6-7.7 (1 H, m),
7.7-7.9 (5 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, DMSO-d6) δ 36.5, 56.8, 119.0, 120.5, 124.5, 125.0, 125.8, 126.1, 129.9, 130.7, 131.0, 131.9, 134.2, 145.4,
149.5, 152.9, 158.3, 176.8, 182.6; EI-MS: 454 (100) [M+], 326 (51), 251 (100); Anal. calc. for C20H11F5N2O3S (454.37): C 52.87, H 2.44, N 6.17; found: C 52.72, H
2.34, N 6.11.
2-Amino-4-(3-cyanophenyl)-5,10-dihydro-5,10-dioxo-4H-benzo[g]chromene-3-carbonitrile (2m). 3-Cyanobenzaldehyde (131 mg, 1.0 mmol) and
malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (3 mL) and three drops of Et3N were added. The reaction mixture was stirred at room temperature
for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The formed
precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 110 mg (0.31 mmol, 31%); red solid of m.p. 257°C; IR: 3425, 3329,
3284, 3256, 3222, 3190, 2235, 2201, 1668, 1656, 1637, 1602, 1589, 1484, 1411, 1370, 1336, 1301, 1243, 1195, 1180, 1160, 1140, 1091, 1074, 7036, 1022, 955, 928,
815, 774, 756, 727, 716, 696; 1H-NMR (300 MHz, DMSO-d6) δ 4.73 (1 H, s), 7.41 (2 H, s), 7.5-7.6 (1 H, m), 7.7-7.8 (2 H, m), 7.8-7.9 (4 H, m), 8.0-8.1 (1 H, m); 13C-
NMR (75.5 MHz, DMSO-d6) δ 36.2, 56.8, 111.6, 118.7, 119.1, 120.4, 125.8, 126.0, 129.7, 130.8, 130.9, 131.4, 132.8, 134.1, 134.5, 145.3, 149.6, 158.3, 176.8, 182.6;
EI-MS: 353 (94) [M+], 251 (100); Anal. calc. for C21H11N3O3 (353.34): C 71.39, H 3.14, N 11.89; found: C 71.31, H 3.22, N 11.78.
2-Amino-4-(4-cyanophenyl)-5,10-dihydro-5,10-dioxo-4H-benzo[g]chromene-3-carbonitrile (2n). 4-Cyanobenzaldehyde (131 mg, 1.0 mmol) and
malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (3 mL) and three drops of Et3N were added. The reaction mixture was stirred at room temperature
for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The formed
precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 160 mg (0.45 mmol, 45%); red solid of m.p. 279-280°C; IR: 3402,
3322, 3284, 3249, 3218, 3194, 2231, 2203, 1675, 1662, 1639, 1602, 1505, 1411, 1363, 1340, 1331, 1303, 1244, 1203, 1178, 1159, 1095, 1074, 1037, 1020, 949, 842,
797, 777, 752, 740, 721, 679; 1H-NMR (300 MHz, DMSO-d6) δ 4.74 (1 H, s), 7.44 (2 H, s), 7.56 (2 H, dd, J = 8.4 Hz, 1.8 Hz), 7.78 (2 H, dd, J = 8.4 Hz, 1.8 Hz), 7.8-
7.9 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz, DMSO-d6) δ 36.6, 56.5, 109.9, 118.7, 119.0, 120.7, 125.8, 126.1, 128.9, 130.7, 130.9, 132.6, 134.2, 134.5,
149.0, 149.5, 158.4, 176.7, 182.5; EI-MS: 353 (95) [M+], 251 (100); Anal. calc. for C21H11N3O3 (353.34): C 71.39, H 3.14, N 11.89; found: C 71.29, H 3.20, N 11.81.
2-Amino-5,10-dihydro-5,10-dioxo-4-(4-ethynylphenyl)-4H-benzo[g]chromene-3-carbonitrile (2o). 4-Ethynylbenzaldehyde (130 mg, 1.0 mmol) and
malononitrile (70 mg, 1.0 mmol) were dissolved in MeCN (3 mL) and three drops of Et3N were added. The reaction mixture was stirred at room temperature
for 30 min. 2-Hydroxy-1,4-naphthoquinone (174 mg, 1.0 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The formed
precipitate was collected, washed with MeCN and n-hexane and dried in vacuum. Yield: 114 mg (0.32 mmol, 32%); orange-red solid of m.p. > 395°C; IR:
3401, 3322, 3296, 3215, 2193, 1693, 1662, 1639, 1601, 1593, 1529, 1505, 1414, 1365, 1336, 1301, 1241, 1208, 1182, 1161, 1095, 1073, 1042, 1018, 963, 947, 849,
799, 779, 755, 744, 720, 693; 1H-NMR (300 MHz, DMSO-d6) δ 4.14 (1 H, s), 4.64 (1 H, s), 7.3-7.4 (6 H, m), 7.8-7.9 (3 H, m), 8.0-8.1 (1 H, m); 13C-NMR (75.5 MHz,
DMSO-d6) δ 57.0, 80.8, 83.2, 119.2, 120.5, 121.4, 125.8, 126.1, 128.1, 130.7, 131.0, 131.9, 134.2, 134.5, 144.5, 149.1, 158.4, 176.8, 182.6; EI-MS: 352 (100) [M+],
251 (81); Anal. calc. for C22H12N2O3 (352.35): C 74.99, H 3.43, N 7.95; found: C 75.10, H 3.31, N 7.82.
7
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10.1002/cbdv.202000839
Chemistry & Biodiversity
Chem. Biodiversity
Toxoplasma gondii cell line, culture conditions, and assay
Vero cells (ATCC® CCL81™, USA) were applied for the cultivation of T. gondii tachyzoites of the RH strain. The Vero cells in 96-Well plates were cultured in
RPMI 1640 medium (5 x 103 cells/well in 200 µL) with 10% heat inactivated fetal bovine serum (FBS, Invitrogen, USA) in a humidified 5% CO2 atmosphere
at 37°C. The inhibition assay with the T. gondii parasites in Vero cells was carried out as described previously.[25,39]
Leishmania major cell isolation and culture conditions
Promastigotes of L. major were isolated in February 2016 and maintained in Schneider’s Drosophila medium (Invitrogen, USA), which was supplemented
with 10% FBS and antibiotics, in a tissue culture flask with weekly transfers at 26°C. Promastigotes were cryopreserved in liquid nitrogen at concentrations
of 3 × 106 parasite/mL. The virulence of L. major parasites was maintained by passing in female BALB/c mice via injection of 1 x 106 stationary-phase
promastigotes in the hind footpads. L. major amastigotes were isolated from the treated mice after 8 weeks. The transformation of the isolated amastigotes
into promastigotes was achieved by culturing at 26°C in Schneider's medium supplemented with 10% FBS and antibiotics. Amastigote-derived
promastigotes with less than five in vitro passages were used for the infection of mice. Male and female BALB/c mice were obtained from Pharmaceutical
College, King Saud University, Saudi Arabia, and maintained in specific pathogen-free facilities.[40,41] Concerning the mentioned laboratory animals, we
followed all the instructions and rules designed by the Scientific Research Deanship, Qassim University, Saudi Arabia, under the permission/accreditation
number 10124-cosao-2020-1-3-I.
Leishmania major cell assays
Cell assays with L. major promastigotes and amastigotes were carried out as described previously.[41-48]
Trypanosoma cell line and culture conditions
Cultivation of the T. b. brucei bloodstream form cell strain Lister 427 was carried out in HMI-9 medium, pH 7.5, supplemented with 10% FBS at 37°C in a
humidified 5% CO2 atmosphere.[3,42]
Alamar Blue (AB) assay
Viable cells after treatment with drug candidates were identified via the AB assay.[42-45] Pink resofurin is formed in intact cells from the irreversible reaction
of the blue dye resazurin and NADH. T. b. brucei cells (8000/well) were seeded on 96-well microplates, test compounds (dissolved in DMSO) were added
and the cells were incubated for 72 h (5% CO2, 95% humidity, 37°C). AB reagent (10 µL of 500 µM resazurin sodium salt in PBS) was added and the cells
were incubated for additional 4 h at 37°C. Fluorescence (extinction at 544 nm, emission at 590 nm) was determined on an Omega Fluostar (BMG Labtech)
fluorescence plate reader. The IC50 values were determined with the Quest GraphTM IC50 Calculator (AAT Bioquest Inc.).[47]
In vitro cytotoxicity assay
MTT assay with Vero cells was carried out for cytotoxicity evaluation of compounds. The assay was carried out as reported previously.[47,48]
Antioxidant assay
Aliquots of eight concentrations (1, 5, 10, 20, 25, 50, 100, 150 µM) of test compounds dissolved in methanol were added to eight test tubes. Compounds
were accurately dissolved in methanol to achieve the required concentrations by dilution techniques. 5 mL of 0.004% 1,1-diphenyl-2-picrylhydrazil (DPPH)
solution was given to each test tube using a micropipette. The solutions were kept at room temperature for 30 minutes to complete the reaction. DPPH
was added to a blank test tube containing only methanol. After 30 min the absorbance was measured with a double beam spectrophotometer (JASCO V-
630) at 517nm. IC50 values were calculated from the plot of inhibition (In %) vs. concentration (in µM).[46,47] The free radical scavenging kinetics for standard
antioxidant viz. ascorbic acid and the test compounds were calculated using the following formula:
DPPH scavenging effect (%) = {Acontrol – Asample / Acontrol} x 100;
The biological activities of the ligands were examined in terms of DPPH radical scavenging activities where the count fraction causing 50% inhibition of
DPPH is called IC50.[49,50]
8
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Cyclovoltammetry
Cyclic voltammograms were recorded of test compounds dissolved in DMSO. Bioanalytical System BASi EPSILON Model instrument with X–Y recorder
was applied with a three-electrode framework consisting of a glassy carbon working anode, Ag+/AgCl as reference electrode, a platinum wire as auxiliary
electrode in 0.1 M Et4NClO4 as supporting electrode. Ferrocene served as internal standard.[27] The direction of feed potential was from anode to cathode.
Supplementary Material
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/MS-number.
Acknowledgements
K. A. was supported by the MCE Society, Pune, India, for a research stay at Organic Chemistry 1, University of Bayreuth, Germany.
Author Contribution Statement
B. B. prepared the compounds and wrote the article. I. S. N., T. A. K. and J. J. carried out the antiparasitic assays. A. S. and P. S. S. carried out the antioxidant
assay and the cyclovoltammetry experiments. W. S. K., K. A., K. E. and R. S. provided the material, supervised the work and proofread the article.
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Twitter Text
New anti-parasitic pyranonaphthoquinone derivatives were prepared which were active against Toxoplasma gondii, Trypanosoma brucei,
and Leishmania major parasites.
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