New Antiparasitic Bis-Naphthoquinone Derivatives

Article abstract of DOI:10.1002/cbdv.201900597

A series of bis-naphthoquinone derivatives prepared by condensation of aryl aldehydes with lawsone were tested for antiparasitic activities against Toxoplasma gondii and Trypanosoma brucei parasites. Monofluorophenyl derivative 1a, 3,4-difluorophenyl anal

Full text of DOI:10.1002/cbdv.201900597

Title: New anti-parasitic bis-naphthoquinone derivatives
Authors: Bernhard Biersack, Jana Jentzsch, Waleed S. Koko, Ibrahim
S. Al Nasr, Tariq A. Khan, Rainer Schobert, and Klaus Ersfeld
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10.1002/cbdv.201900597
Chemistry & Biodiversity
Chem. Biodiversity
New anti-parasitic bis-naphthoquinone derivatives
Jana Jentzsch,a Waleed S. Koko,b Ibrahim S. Al Nasr,b,c Tariq A. Khan,d Rainer Schobert,e Klaus Ersfeld,a
and Bernhard Biersack*,e
aLaboratory of Molecular Parasitology, University of Bayreuth, Universitätsstrasse 30, 95440 Bayreuth, Germany
bCollege of Science and Arts in Ar Rass, Qassim University, King Abdelaziz Road, Ar Rass 51921, Saudi Arabia
cCollege of Science and Arts in Unaizah, Qassim University, Unaizah 51911, Saudi Arabia
dCollege of Applied Health Sciences in Ar Rass, Qassim University, Ar Rass 51921, Saudi Arabia
eOrganic Chemistry Laboratory, University of Bayreuth, Universitätsstrasse 30, 95440 Bayreuth, Germany, e-mail bernhard.biersack@yahoo.com
A series of bis-naphthoquinone derivatives prepared by condensation of aryl aldehydes with lawsone was tested for anti-parasitic activities
against Toxoplasma gondii and Trypanosoma brucei parasites. Monofluorophenyl derivative 1a, 3,4-difluorophenyl analog 1c and furyl compound
1l exhibited significant activity against T. gondii cells and appear to be new promising drug candidates against this parasite. The 3,4,5-
trifluorophenyl derivative 1g and the isovanillyl derivative 1j displayed selective activity against Leishmania major amastigotes.
Keywords: lawsone • neglected tropical diseases • fluorine • furan
Introduction
Parasitic diseases still represent an enormous world-wide medical challenge.[1] Neglected tropical diseases (NTDs) pose a particularly great danger
both for local people and travelers in many tropical and subtropical countries.[2] Due to the climate change further regions of the world will likely
be affected by these parasitic diseases in the future.[2] There are only a few drugs available against NTDs because big pharma is reluctant to
develop new and more potent drugs against diseases mainly poor people suffer from.[3] Vice versa, poor people affected by NTDs are in want of
affordable drugs, which could, however, arise from tapping into natural resources. Many natural product derived drug candidates, active against
various NTDs, have emerged.[3] In addition, natural products constitute sustainable, cheap materials.
Lawsone, for instance, is a natural 2-hydroxy-1,4-naphthoquinone isolated from the Henna plant (Lawsonia inermis) which is being applied for
the management of skin diseases in South Asia as a component of the local Ayurveda and Unani folk medicine.[4,5] Lawsone / 2-hydroxy-1,4-
naphthoquinone is also a useful starting material for the preparation of various related quinones with proven bioactivity such as lapachol or
atovaquone.[4,5] Arylmethylene-substituted bis-naphthoquinone derivatives have been reported to exhibit only moderate activities against human
cancer cells which makes them interesting candidates for the design of selective anti-parasitic drugs.[6] Indeed, a small series of substituted bis-
2-hydroxy-1,4-naphthoquinones including 1a (Figure 1) was obtained with distinct activity against Leishmania parasites.[7] Herein, we report on
expanded series of such new derivatives (e.g., fluoroarenes, vanillins, furans) with optimized activity against and selectivity for various parasites
including Toxoplasma gondii, Trypanosoma brucei and Leishmania.
O
O
O
O
O
OH
OH HO
(i)
O
Ar
Lawsone
1
Scheme 1. Reagents and conditions: (i) Aryl aldehyde (0.5 equiv.), cat. β-alanine, AcOH, 50 °C, 2 h, 30-62%.
1
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10.1002/cbdv.201900597
Chemistry & Biodiversity
Chem. Biodiversity
Results and Discussion
The known compounds 1a–b and the new analogs 1c–l were obtained as solids from the one-pot condensation reaction of two equivalents of
2-hydroxy-1,4-naphthoquinone (lawsone) with the corresponding aryl aldehyde and a catalytic amount of β-alanine in warm acetic acid (Scheme
1).[7,8] The structures of the target compounds 1a–l are shown in Figure 1.
O
O
HO
OH
O
O
Ar
1
Ar:
C
F
C
F
C
F
F
c
a
b
C
C
F
C
F
F
F
F
d
e
f
F
F
F
F
C
C
F
F
F
F
g
h
F
F
C
C
OH
OMe
j
OMe
i
OH
C
C
O
O
l
k
Figure 1. Structures of target compounds 1a–l.
The anti-parasitic activity of the compounds 1a–l was initially tested against Toxoplasma gondii and Trypanosoma brucei parasites and compared
with the toxicity against normal kidney epithelial Vero cells (Table 1). The best performing compounds 1a and 1c exhibited good and similar
activities against T. gondii cells with 1c showing a better selectivity for T. gondii cells over Vero cells. Like 1a, the pentafluorophenyl derivative 1h
was more toxic to normal Vero cells than to T. gondii cells. Atovaquone, which is commonly applied for the treatment of toxoplasmosis, was used
as positive control and displayed the highest activity and selectivity.[9] However, the toxicity of most bis-naphthoquinones (except for 1a and 1h)
to Vero cells was distinctly lower when compared with atovaquone. In contrast to this varied spectrum of activities against T. gondii, all compounds,
except 1a and 1i which displayed low double-digit IC50 values, were virtually inactive against T. b. brucei cells. Pentamidine, an approved drug for
the treatment of sleeping sickness, was applied as positive control with activity in the nano-molar concentration range.[10]
The compounds 1a–l were also tested against Leishmania major promastigotes and amastigotes in comparison to normal Vero cells (Table 2).
Compounds 1a and 1h showed similar moderate activities against the promastigotes (EC50 = 35.0 µM for 1a and 35.5 µM for 1h). Compounds
1c and 1f were most active against the amastigotes (EC50 = 23.7 µM for 1c and 22.6 µM for 1f) and more active than the known compound 1a.
However, these compounds showed only poor selectivity. In contrast to that, compounds 1g and 1j displayed only slightly weaker activities than
2
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10.1002/cbdv.201900597
Chemistry & Biodiversity
Chem. Biodiversity
1c and 1f against the amastigotes (EC50 = 34.1 µM for 1g and 37.9 µM for 1j) but reasonable selectivities when compared with their effects on
Vero cells (SI = 4.18 for 1g and more than 2.64 for 1j). The activity of the known compound 1a against L. major promastigotes (EC50 = 35.0 µM)
was much lower when compared with its published activities against L. amazonensis (IC50 = 0.6 µM) and L. braziliensis promastigotes (IC50 = 0.8
µM).[7] Amphotericin B was applied as a proper positive control for the tests with L. major parasites despite of its severe side-effects.[11] In this
study, the toxicity of amphotericin B to Vero cells was distinctly higher when compared with 1g and 1j , for example, which did not harm Vero
cells at doses of 100 µM and still showed moderate activity against amastigotes.
Table 1. Effective concentrations EC50 of compounds 1a–l when applied to cells of Toxoplasma gondii and their inhibitory concentrations IC50 when applied to
Trypanosoma brucei brucei cells as well as IC50 values for Vero (African green monkey kidney epithelial) cells.[a] Amphotericin B (AmB) , pentamidine, and atovaquone
(ATO) were applied as positive controls.
Compd.
IC50 (Vero,
µM)
EC50 (T.
SI (Vero / T.
gondii)[b]
IC50 (T. b.
SI (Vero / T. b.
brucei)[c]
gondii, µM)
brucei), µM)
1a
6.78
11.64
26.63
13.95
34.53
27.94
23.86
48.94
21.47
109.0
59.28
25.33
23.22
-
0.58
2.0
1.26
0.80
1.41
1.01
2.91
0.32
-
15.6
> 10
> 10
> 100
> 10
> 10
-
0.43
1b
53.48
17.63
27.54
39.37
24.06
142.3
6.88
-
1c
-
1d
-
1e
-
1f
-
1g
-
1h
72.3
27.8
-
0.10
1i
> 100
> 100
57.41
68.25
7.7
> 3.60
1j
-
-
-
-
-
.
1k
2.27
2.94
-
> 100
> 10
-
1l
AmB
Pentamidine
ATO
-
-
-
0.042[d]
9.5
0.07
136
-
-
[a] Values are the means of at least three independent experiments (SD ± 15%). They were derived from concentration–response curves obtained by measuring the
[b]
percentage of vital cells relative to untreated controls after 72 h.
Selectivity index (EC50/IC50) calculated from the corresponding EC50 (T. gondii) and IC50 (Vero)
values. [c] Selectivity index calculated from the corresponding IC50 (T. b. brucei) and IC50 (Vero) values. [d] Value is taken from ref. 8.
Table 2 Effective concentrations EC50 of compounds 1a-l when applied to promastigotes and amastigotes of Leishmania major.* Amphotericin B (AmB) was applied
as positive control.
Compd.
EC50 (promastigotes,
µM)
EC50 (amastigotes,
µM)
SI (Vero /
SI (Vero /
promastigotes)†
amastigotes)‡
1a
1b
1c
1d
1e
1f
35.0
116.0
92.5
89.1
83.0
76.5
108
32.4
48.0
23.7
77.2
36.4
22.6
34.1
30.1
106.0
37.9
33.5
53.7
0.47
0.19
0.46
0.19
0.31
0.48
0.32
1.32
0.19
-
0.21
1.12
0.74
0.36
1.08
1.06
4.18
0.23
>0.94
>2.64
1.71
0.73
16.4
1g
1h
1i
35.5
>200
97.4
101
1j
>1.03
0.57
1.27
9.6
1k
1l
94.1
0.83
AmB
*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. †Selectivity index (IC50/EC50) calculated from the corresponding IC50 values for Vero cells and the EC50
values against L. major promastigotes. ‡Selectivity index calculated from the corresponding IC50 values for Vero cells and the EC50 values for L. major amastigotes.
3
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10.1002/cbdv.201900597
Chemistry & Biodiversity
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Conclusions
A series of bis-naphthoquinone derivatives with variance in the aryl residue at the bridging methylene carbon were prepared and tested on
various protozoal parasites against which they showed distinctly different activities. We identified compounds that were remarkably active against
T. gondii cells (e.g., the fluorophenyl derivatives 1a and 1c, and the furan 1l), and sufficiently selective for L. major amastigotes (e.g., the
fluorophenyl derivative 1g and isovanillyl derivative 1j) to warrant further drug optimization studies. It should be noted that in our tests with
different parasites we could not confirm the excellent activities published for 1a.[7] Although this compound was among the most active ones of
this series, its toxic effect on normal epithelial Vero cells is unfavorable for an anti-parasitic drug. It might hold, however, like the new compound
1h, some potential as an anti-tumoral drug candidate.
Experimental Section
Chemistry
All starting compounds were purchased from Aldrich. The known compounds 1a and 1b were prepared according to literature procedures.[7,12]
The following instruments were used: melting points (uncorrected), Gallenkamp; IR spectra, Perkin-Elmer Spectrum One FT-IR spectrophotometer
with ATR sampling unit; nuclear magnetic resonance spectra, BRUKER Avance 300 spectrometer; chemical shifts are given in parts per million (d)
downfield from tetramethylsilane as internal standard; mass spectra, Varian MAT 311A (EI), Q Exactive (ESI, HRMS, solutions of MeCN);
microanalyses, Perkin-Elmer 2400 CHN elemental analyzer. All tested compounds are >95% pure by elemental analysis.
3,3’-(4-Fluorophenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1a)
Yield: 69 mg (0.15 mmol, 30%); m/z (%) 454 (21) [M+*], 436 (21), 408 (31), 252 (100), 224 (22), 196 (27), 174 (63), 105 (68), 76 (46).
3,3’-(3-Fluorophenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1b)
Yield: 68 mg (0.15 mmol, 30%); m/z (%) 454 (83) [M+*], 436 (57), 408 (84), 280 (33), 251 (100), 174 (70), 105 (87), 76 (56).
3,3’-(3,4-Difluorophenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1c) – General procedure
2-Hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), 3,4-difluorobenzaldehyde (71 mg, 0.5 mmol) and a catalytic amount of β-alanine (15 mg)
were dissolved in AcOH (8 mL). The reaction mixture was stirred at 50 °C for 2 h. The reaction mixture was poured on water (30 mL), the precipitate
was collected and dried in vacuum. Yield: 70 mg (0.15 mmol, 30%); yellow solid of m.p. 109-110°C; υmax(ATR)/cm-1 3340, 1642, 1593, 1515, 1460,
1431, 1362, 1336, 1269, 1211, 1162, 1115, 1044, 1013, 977, 918, 887, 868, 816, 756, 723, 693; 1H NMR (300 MHz, CDCl3)  6.10 (1 H. s), 7.0-7.2 (3
H, m), 7.6-7.8 (4 H, m), 8.0-8.1 (4 H, m); 13C NMR (75.5 MHz, CDCl3)  37.4 (CH), 110.7 (quinone-C3), 116.7-117.6 (m, fluoroaryl-C2, C5), 122.0
(fluoroaryl-C6), 124.3, 126.4, 126.5, 126.7, 127.3, 129.4, 132.7, 133.3 (aryl-carbons), 135.3-135.6 (m, fluoroaryl-C1), 147.6 (fluoroaryl-C3), 154.3 (C-
OH), 181.3 (CO), 184.1 (CO); m/z (%) 472 (63) [M+*], 454 (55), 426 (72), 270 (100), 242 (22), 214 (36), 174 (86), 105 (86) 76 (52); HRMS (ESI) calcd
for C27H15O6F2, [M + H]+, 473.08312; Found 473.08213.
3,3’-(3,5-Difluorophenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1d)
Compound 1d was prepared analogously to 1c from 2-hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), 3,5-difluorobenzaldehyde (71 mg,
0.5 mmol) and a catalytic amount of β-alanine (15 mg) in AcOH (8 mL). Yield: 90 mg (0.21 mmol, 42%); yellow solid of m.p. 116-118°C;
υmax(ATR)/cm-1 3327, 1649, 1624, 1594, 1459, 1365, 1337, 1300, 1266, 1213, 1158, 1115, 1045, 990, 919, 853, 795, 725, 686; 1H NMR (300 MHz,
CDCl3)  6.14 (1 H, s), 6.6-6.7 (1 H, m), 6.7-6.8 (2 H, m), 7.6-7.8 (4 H, m), 8.0-8.1 (4 H, m); 13C NMR (75.5 MHz, CDCl3)  37.6 (CH), 101.8-102.5 (m,
fluoroaryl-C4), 110.7 (quinone-C3), 111.1-111.5 (m, fluoroaryl-C2, C6), 121.4, 126.4, 126.7, 127.3, 129.4, 132.7, 133.1, 133.3, 135.3 (aryl carbons),
142.7-142.9 (m, fluoroaryl-C1), 154.5 (C-OH), 161.2-164.6 (m, fluoroaryl-C3, C5), 181.3 (CO), 184.0 (CO); m/z (%) 472 (92) [M+*], 454 (69), 426
(100), 382 (23), 359 (29), 326 (18), 298 (31), 269 (31), 214 (23), 105 (62), 104 (61), 76 (38); HRMS (ESI) calcd for C27H15O6F2, [M + H]+, 473.08312;
Found 473.08267.
3,3’-(2,4-Difluorophenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1e)
Compound 1e was prepared analogously to 1c from 2-hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), 2,4-difluorobenzaldehyde (71 mg, 0.5
mmol) and a catalytic amount of β-alanine (15 mg) in AcOH (8 mL). Yield: 138 mg (0.29 mmol, 58%); yellow solid of m.p. 169-170°C; υmax(ATR)/cm-
1 3336, 1647, 1593, 1502, 1461, 1428, 1364, 1338, 1297, 1274, 1258, 1211, 1161, 1139, 1090, 1044, 1012, 967, 938, 903, 848, 816, 798, 782, 722,
4
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10.1002/cbdv.201900597
Chemistry & Biodiversity
Chem. Biodiversity
693, 679, 646, 608; 1H NMR (300 MHz, CDCl3)  6.23 (1 H, s), 6.7-6.8 (2 H, m), 7.1-7.2 (1 H, m), 7.6-7.8 (4 H, m), 8.0-8.1 (4 H, m); 13C NMR (75.5
MHz, CDCl3)  32.1 (CH), 103.1-103.6 (m, fluoroaryl-C3), 110.5-110.8 (m, fluoroaryl-C5, quinone-C3), 121.3, 126.3, 127.2, 129.3, 130.2-130.5 (m),
132.8, 133.1, 135.2 (aryl carbons), 154.0 (C-OH), 161.0-164.0 (m, fluoroaryl-C2, C4), 181.3 (CO), 183.8 (CO); m/z (%) 472 (4) [M+*], 452 (100), 426
(53), 279 (27), 251 (32), 174 (26), 105 (60), 76 (48); HRMS (ESI) calcd for C27H15O6F2, [M + H]+, 473.08312; Found 473.08247.
3,3’-(2,4,5-Trifluorophenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1f)
Compound 1f was prepared analogously to 1c from 2-hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), 2,4,5-trifluorobenzaldehyde (80 mg,
0.5 mmol) and a catalytic amount of β-alanine (15 mg) in AcOH (8 mL). Yield: 130 mg (0.27 mmol, 54%); yellow solid of m.p. 167-169°C;
υmax(ATR)/cm-1 3347, 1647, 1593, 1513, 1461, 1424, 1364, 1334, 1276, 1261, 1212, 1188, 1151, 1098, 1043, 1011, 970, 938, 911, 870,3 840, 816,
796, 762, 723, 688, 658, 638; 1H NMR (300 MHz, CDCl3)  6.20 (1 H, s), 6.8-6.9 (1 H, m), 7.0-7.1 (1 H, m), 7.6-7.8 (4 H, m), 8.0-8.1 (4 H, m);13C NMR
(75.5 MHz, CDCl3)  32.0 (CH), 104.9-105.3 (m, fluoroaryl-C3), 117.8-118.1 (m, fluoroaryl-C6), 120.7 (fluoroaryl-C6), 126.4, 127.3, 129.2, 132.8,
133.2, 135.3 (aryl carbons), 148.0 (fluoroaryl-C5), 151.0 (fluoroaryl-C4), 153.9 (C-OH), 157.5 (fluoroaryl-C2), 181.2 (CO), 183.6 (CO); m/z (%) 490
(9) [M+*], 470 (100), 444 (76), 298 (37), 288 (31), 269 (47), 104 (72 ), 76 (54); HRMS (ESI) calcd for C27H14O6F3, [M + H]+, 491.07370; Found: 491.07311.
3,3’-(3,4,5-Trifluorophenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1g)
Compound 1f was prepared analogously to 1c from 2-hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), 3,4,5-trifluorobenzaldehyde (80 mg,
0.5 mmol) and a catalytic amount of β-alanine (15 mg) in AcOH (8 mL). Yield: 120 mg (0.25 mmol, 50%); yellow solid of m.p. 116-117°C;
υmax(ATR)/cm-1 3318, 1647, 1620, 1593, 1527, 1460, 1444, 1363, 1338, 1260, 1213, 1159, 1040, 998, 971, 919, 879, 868, 852, 817, 797, 725, 700; 1H
NMR (300 MHz, CDCl3)  6.08 (1 H, s), 6.9-7.0 (2 H, m), 7.7-7.8 (4 H, m), 8.1-8.2 (4 H, m); 13C NMR (75.5 MHz, CDCl3)  37.5 (CH), 110.7 (quinone-
C3), 112.4-112.7 (m, fluoroaryl-C2, C6), 121.3, 126.4, 126.5, 126.7, 127.3, 129.3, 132.7, 133.1, 133.3, 135.3 (aryl carbons), 148.9 (fluoroaryl-C4),
152.5 (fluoroaryl-C3, C5), 154.3 (C-OH), 181.3 (CO), 183.9 (CO); m/z (%) 490 (62) [M+*], 472 (51), 444 (100), 416 (11), 400 (14), 359 (27), 344 (12),
288 (26), 232 (14), 174 (20), 147 (12), 104 (38), 76 (21); HRMS (ESI) calcd for C27H14O6F3, [M + H]+, 491.07370; Found: 491.07262.
3,3’-(2,3,4,5,6-Pentafluorophenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1h)
Compound 1h was prepared analogously to 1c from 2-hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), pentafluorobenzaldehyde (98 mg,
0.5 mmol) and a catalytic amount of β-alanine (15 mg) in AcOH (6 mL). Yield: 160 mg (0.30 mmol, 61%); yellow solid of m.p. 110-111°C;
υmax(ATR)/cm-1 3327, 1648, 1594, 1522, 1499, 1364, 1339, 1275, 1213, 1117, 1045, 999, 977, 911, 785, 724, 699; 1H NMR (300 MHz, CDCl3)  6.17
(1 H, s), 7.59 (2 H, s), 7.6-7.8 (4 H, m), 8.0-8.1 (4 H, m); 13C NMR (75.5 MHz, CDCl3)  29.1 (CH), 113.8-114.2 (m, fluoroaryl-C1), 119.7, 126.4, 126.9,
127.2, 129.2, 132.7, 133.2, 135.4 (aryl carbons), 137.8-139.2 (m, fluoroaryl-C3, C5), 141.5-142.2 (m, fluoroaryl-C2, C6), 143.7-144.6 (m, fluoroaryl-
C2, C6), 147.1-148.6 (m, fluoroaryl-C4), 153.4 (C-OH), 181.1 (CO), 183.4 (CO); m/z (%) 526 (79) [M+*], 506 (66), 480 (100) 434 (20), 359 (36), 334
(64), 305 (51), 174 (38), 105 (78), 104 (79), 76 (47); HRMS (ESI) calcd for C27H12O6F5, [M + H]+, 527.05486; Found 527.05347.
3,3’-(4-Hydroxy-3-methoxyphenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1i)
Compound 1i was prepared analogously to 1c from 2-hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), vanillin (76 mg, 0.5 mmol) and a
catalytic amount of β-alanine (15 mg) in AcOH (6 mL). Yield: 80 mg (0.17 mmol, 34%); yellow solid of m.p. 107-108°C; υmax(ATR)/cm-1 3294, 1645,
1592, 1580, 1513, 1460, 1431, 1362, 1336, 1252, 1206, 1155, 1122, 1030, 972, 915, 867, 813, 796, 722; 1H NMR (300 MHz, CDCl3)  3.79 (3 H, s),
5.52 (1 H, s), 6.7-6.8 (3 H, m), 7.6-7.8 (4 H, m), 7.9-8.1 (6 H, m); 13C NMR (75.5 MHz, CDCl3)  37.6 (CH), 56.0 (MeO), 110.7 (quinone-C3), 111.3
(phenyl-C2), 114.1 (phenyl-C5), 121.1 (phenyl-C6), 122.9, 126.3, 126.5, 127.2, 129.6, 132.8, 133.2, 135.1 (aryl carbons), 144.6 (phenyl-C4), 146.5
(phenyl-C3), 154.5 (quinone-C-OH), 181.4 (CO), 184.6 (CO); m/z (%) 482 (8) [M+*], 464 (5), 308 (45), 280 (100), 263 (14), 249 (27), 237 (19), 209
(12), 174 (73), 146 (16), 105 (64), 76 (27); HRMS (ESI) calcd for C28H19O8, [M + H]+, 483.10744; Found 483.10624.
3,3’-(3-Hydroxy-4-methoxyphenylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1j)
Compound 1j was prepared analogously to 1c from 2-hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), isovanillin (76 mg, 0.5 mmol) and a
catalytic amount of β-alanine (15 mg) in AcOH (6 mL). Yield: 80 mg (0.17 mmol, 34%); yellow solid of m.p. 114-116°C; υmax(ATR)/cm-1 3334, 1646,
1592, 1579, 1510, 1460, 1441, 1362, 1336, 1259, 1211, 1128, 1045, 1023, 981, 916, 885, 868, 798, 740, 723, 692, 656; 1H NMR (300 MHz, CDCl3) 
3.83 (3 H, s), 5.53 (1 H, s), 6.14 (1 H, s), 6.3-6.4 (3 H, m), 7.6-7.8 (4 H, m), 8.0-8.1 (4 H, m);13C NMR (75.5 MHz, CDCl3)  37.0 (CH), 55.9 (MeO), 110.4
(quinone-C3), 114.6 (phenyl-C5), 119.6 (phenyl-C2), 122.7 (phenyl-C6), 126.3, 126.5, 127.2, 129.6, 131.0, 132.7, 133.2, 135.0 (aryl carbons), 145.5
(phenyl-C4), 154.8 (quinone-C-OH), 181.3 (CO), 184.7 (CO); m/z (%) 482 (5) [M+*], 308 (47), 280 (100), 265 (22), 249 (27), 237 (25), 209 (17), 174
(87), 146 (23), 105 (97), 76 (42); HRMS (ESI) calcd for C28H19O8, [M + H]+, 483.10744; Found 483.10706.
3,3’-(2-Furylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1k)
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Compound 1k was prepared analogously to 1c from 2-hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), furfural (57 mg, 0.5 mmol) and a
catalytic amount of β-alanine (15 mg) in AcOH (6 mL). Yield: 80 mg (0.19 mmol, 38%); yellow solid of m.p. 118-120°C; υmax(ATR)/cm-1 3329, 1647,
1592, 1580, 1501, 1460, 1363, 1336, 1261, 1212, 1160, 1142, 1092, 1074, 1043, 1011, 970, 883, 869, 819, 797, 761, 722; 1H NMR (300 MHz, CDCl3)
 6.09 (1 H, d, J = 3.3 Hz), 6.20 (1 H, s), 6.2-6.3 (1 H, m), 7.3-7.4 (1 H, m), 7.6-7.8 (4 H, m), 8.0-8.1 (4 H, m), 8.2-8.3 (2 H, br s); 13C NMR (75.5 MHz,
CDCl3)  31.8 (CH), 107.2 (furyl-C3), 110.5 (quinone-C3), 120.7, 126.4, 127.2, 129.6, 132.6, 133.3, 135.0 (aryl carbons), 141.8 (furyl-C5), 150.5 (furyl-
C2), 154.8 (C-OH), 181.2 (CO), 184.3 (CO); m/z (%) 426 (43) [M+*], 224 (42), 174 (100), 146 (22), 105 (78), 77 (23); HRMS (ESI) calcd for C25H15O7,
[M + H]+, 427.08123; Found 427.08009.
3,3’-(3-Furylmethylene)-bis(2-hydroxy-1,4-naphthalenedione) (1l)
Compound 1l was prepared analogously to 1c from 2-hydroxy-1,4-naphthoquinone (174 mg, 1.00 mmol), furyl-3-aldehyde (57 mg, 0.5 mmol)
and a catalytic amount of β-alanine (15 mg) in AcOH (6 mL). Yield: 100 mg (0.24 mmol, 48%); yellow solid of m.p. 167-170°C; υmax(ATR)/cm-1 3328,
1646, 1592, 1579, 1502, 1460, 1362, 1336, 1264, 1219, 1158, 1071, 1044, 1024, 967, 920, 891, 872, 793, 756, 739, 722, 682, 658; 1H NMR (300 MHz,
CDCl3)  6.0-6.1 (1 H, m), 6.3-6.4 (1 H, m), 7.3-7.4 (1 H, m), 7.6-7.8 (4 H, m), 8.0-8.1 (4 H, m); 13C NMR (75.5 MHz, CDCl3)  28.9 (CH), 111.5 (quinone-
C3), 121.9, 122.2, 126.4, 127.1, 129.6, 132.6, 133.3, 135.0 (aryl carbons), 140.2 (furyl-C2), 143.0 (furyl-C5), 154.9 (C-OH), 181.2 (CO), 184.8 (CO); m/z
(%) 426 (14) [M+*], 252 (21), 224 (66), 196 (57), 174 (100), 146 (24), 139 (27), 105 (96), 76 (42); HRMS (ESI) calcd for C25H15O7, [M + H]+, 427.08123;
Found 427.08056.
Toxoplasma gondii culture conditions
Serial passages of the cell line Vero (ATCC® CCL81™, USA) were used for the cultivation of T. gondii tachyzoites of the RH strain (a gift from Dr.
Saeed El-Ashram, State Key Laboratory for Agrobiotechnology, China Agricultural University, Bejing, China). Vero cells were cultured by using
RPMI 1640 medium (Sigma, USA) with heat-inactivated 10% fetal bovine serum (FBS, Invitrogene, USA) in a humidified 5% CO2 atmosphere at
37 °C. 96-Well plates (5 x 103 cells/well in 200 µL RPMI 1640 medium) were used for the cultivation of the Vero cells and then incubated at 37°C
and 5% CO2 for one day, followed by removal of medium and washing the cells with phosphate buffered saline (PBS). Then, RPMI 1640 medium
with 2% FBS containing tachyzoites (RH strain) of T. gondii at a ratio of 5 (parasite) : 1 (Vero cells) was added. After incubation at 37°C and 5%
CO2 for 5 h, cells were washed with PBS and then overlaid with medium containing test compounds or atovaquone (50, 25, 12.5, 6.25, 3.13, 1.65,
and 0.75 µg mL-1).
After incubation at 37 °C and 5% CO2 for 72 h, the cells were stained with 1% toluidine blue after washing with PBS and fixation in 10% formalin.
The cells were examined under an inverted photomicroscope (MCD-400, Leica, Japan) to determine the infection index (number of infected cells
from 200 tested cells) of T. gondii.[13,14]
Leishmania major promastigotes and amastigotes
Promastigotes of L. major were isolated from a Saudi male patient in February 2016 and maintained at 26°C in Schneider’s Drosophila medium
(Invitrogen, USA) supplemented with 10% heat inactivated FBS (Invitrogen, USA) and antibiotics in a tissue culture flask with weekly transfers.
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 by injecting hind footpads with 1x106 stationary-phase promastigotes. After 8 weeks, L. major amastigotes
were isolated from mice. Isolated amastigotes were transformed to promastigote forms by culturing at 26°C in Schneider's medium
supplemented with 10% FBS and antibiotics. For infection, amastigote-derived promastigotes with less than five in vitro passages were used.
Male and female BALB/c mice were obtained from Pharmaceutical College, King Saud University, Kingdom of Saudi Arabia, and maintained in
specific pathogen-free facilities.
In order to evaluate the activity of test compounds against L. major promastigotes, promastigotes from logarithmic-phase cultured in phenol
red-free RPMI 1640 medium (Invitrogen, USA) with 10% FBS were suspended on 96-wells plates to yield 106 cells mL-1 (200 µL/well) after
hemocytometer counting. Compounds were added to obtain the final concentrations (50, 25, 12.5, 6.25, 3.13, 1.65, and 0.75 µg mL-1). Negative
control wells containing cultures with DMSO (1%) and without compound and positive control wells containing cultures with decreasing
concentration of amphotericin B (reference compound, 50, 25, 12.5, 6.25, 3.13, 1.65, and 0.75 µg mL-1) were used. Plates were incubated at 26°C
for 72 h to evaluate the anti-proliferative effect. The number of viable promastigotes were assessed by colorimetric method using the tetrazolium
salt colorimetric assay (MTT). It measures the reduction of the MTT component into an insoluble formazan product. This colored product was
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solubilized by adding detergent solution to lyse the cells. The samples were analysed by using a microplate absorbance spectrophotometer
(xMark, Bio-Rad, USA) at 570 nm.[14]
In order to evaluate the activity of test compounds against amastigotes in macrophages, peritoneal macrophages from female BALB/c mice (6-
8 weeks of age) were collected by aspiration, then 5 x 104 cells/well were seeded on 96-wells plates in RPMI 1640 medium with 10% FBS for 4 h
at 37°C in 5% CO2 atmosphere to promote cell adhesion. Medium was discarded and washed with PBS. 200 µL containing L. major promastigotes
solution (at a ratio of 10 promastigotes : 1 macrophage in RPMI 1640 medium with 10% FBS) was added per well. Plates were incubated for 24
h at 37°C in humidified 5% CO2 atmosphere to allow infection and amastigote differentiation. Then, the infected macrophages were washed
three times with PBS to remove the free promastigotes and overlaid with fresh RPMI 1640 medium containing compounds at final concentrations
(50, 25, 12.5, 6.25, 3.13, 1.65, and 0.75 µg mL-1) and cells were incubated at 37°C in humidified 5% CO2 atmosphere for 72 h. Negative control
containing cultures with DMSO (1%) and without compounds and positive control wells containing cultures with decreasing concentration of
amphotericin B (reference compound, 50, 25, 12.5, 6.25, 3.13, 1.65, and 0.75 µg mL-1) were used. The percentage of infected macrophages were
evaluated microscopically after removing medium, washing, fixation, Giemsa staining.[14]
Trypanosoma cell line and culture conditions
Cells of the T. b. brucei bloodstream trypomastigote cell line Lister 427 were maintained in HMI-9 medium, pH 7.5, supplemented with 10% FBS
in a humidified 5% CO2 atmosphere at 37 °C.[15]
Alamar Blue (AB) assay
The AB assay was used to identify viable cells after treatment with drug candidates.[16-19] This assay bases on the irreversible reaction of the blue
dye resazurin and NADH to pink resofurin in intact cells. T. b. brucei cells (8000/well) were seeded on 96-well microplates, treated with the test
compounds (dissolved in DMSO) and incubated for 72 h (5% CO2, 95% humidity, 37 °C). 10 µL of the AB reagent (500 mM resazurin sodium salt
in PBS) was added and incubated for further 4 h at 37 °C. The fluorescence (extinction at 544 nm, emission at 590 nm) was measured on an
Omega Fluostar (BMG Labtech) fluorescence plate reader.
Cytotoxicity assay
MTT assay was carried out for cytotoxicity evaluation of compounds. Briefly, Vero cells were cultured in 96-well plates (5 × 103 cells/well/200 µL)
for 24 h in RPMI 1640 medium with 10% FBS and 5% CO2 at 37°C. Cells were washed with PBS and treated with test compounds for 72 h at
varying concentrations (50, 25, 12.5, 6.25, 3.13, 1.65, and 0.75 µg mL-1) in medium with 10% FBS. As negative control, cells were treated with
medium only. Thereafter, the supernatant was removed and 50 µL RPMI 1640 medium containing 14 µL MTT (5 mg mL-1) was added and
incubated for 4 h. After that, the supernatant was removed and 150 µL DMSO was added in order to dissolve the formazan. Microplate absorbance
spectrophotometer was applied for colorimetric analysis (λ = 540 nm). Cytotoxic effects were expressed by IC50 values (concentration that caused
a 50% reduction in viable cells).[14,20]
Acknowledgements
The authors gratefully acknowledge Qassim University, represented by the Deanship of Scientific Research on the material support for this
research under the number cosao-2018-1-14-s-3859 during the academic year 1439 AH/2018 AD.
Author Contribution Statement
B. B. prepared the test compounds and wrote the article. I. S. N., T. K. and J. J. carried out the anti-parasitic assays. W. S. K., K. E. and R. S.
provided the material, supervised the work and proofread the article.
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Entry for the Graphical Illustration
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Twitter Text
New bis-naphthoquinone condensation products were identified with improved selectivities for certain protozoal parasites.
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