Novel carbazole aminoalcohols as inhibitors of β-hematin formation: Antiplasmodial and antischistosomal activities

Article abstract of DOI:10.1016/j.ijpddr.2017.03.007

Malaria and schistosomiasis are two of the most socioeconomically devastating parasitic diseases in tropical and subtropical countries. Since current chemotherapeutic options are limited and defective, there is an urgent need to develop novel antiplasmodials and antischistosomals. Hemozoin is a disposal product formed from the hemoglobin digestion by some blood-feeding parasites. Hemozoin formation is an essential process for the parasites to detoxify free heme, which is a reliable therapeutic target for identifying novel antiparasitic agents. A series of novel carbazole aminoalcohols were designed and synthesized as potential antiplasmodial and antischistosomal agents, and several compounds showed potent in?vitro activities against Plasmodium falciparum 3D7 and Dd2 strains and adult and juvenile Schistosoma japonicum. Investigations on the dual antiparasitic mechanisms showed the correlation between inhibitory activity of β-hematin formation and antiparasitic activity. Inhibiting hemozoin formation was identified as one of the mechanisms of action of carbazole aminoalcohols. Compound 7 displayed potent antiplasmodial (Pf3D7 IC₅₀?=?0.248?μM, PfDd2 IC₅₀?=?0.091?μM) and antischistosomal activities (100% mortality of adult and juvenile schistosomes at 5 and 10?μg/mL, respectively) and exhibited low cytotoxicity (CC₅₀?=?7.931?μM), which could be considered as a promising lead for further investigation. Stoichiometry determination and molecular docking studies were also performed to explain the mode of action of compound 7.

Full text of DOI:10.1016/j.ijpddr.2017.03.007

Accepted Manuscript
Novel carbazole aminoalcohols as inhibitors of β-hematin formation: Antiplasmodial
and antischistosomal activities
Weisi Wang, Qiang Li, Yufen Wei, Jian Xue, Xiao Sun, Yang Yu, Zhuo Chen, Shizhu
Li, Liping Duan
PII:
S2211-3207(16)30091-4
10.1016/j.ijpddr.2017.03.007
IJPDDR 182
DOI:
Reference:
To appear in: International Journal for Parasitology: Drugs and Drug
Resistance
Received Date: 23 September 2016
Revised Date: 29 March 2017
Accepted Date: 29 March 2017
Please cite this article as: Wang, W., Li, Q., Wei, Y., Xue, J., Sun, X., Yu, Y., Chen, Z., Li, S.,
Duan, L., Novel carbazole aminoalcohols as inhibitors of β-hematin formation: Antiplasmodial and
antischistosomal activities, International Journal for Parasitology: Drugs and Drug Resistance (2017),
doi: 10.1016/j.ijpddr.2017.03.007.
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Novel carbazole aminoalcohols as inhibitors of β-hematin formation:
antiplasmodial and antischistosomal activities
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Weisi Wang a, b, Qiang Li , Yufen Wei , Jian Xue , Xiao Sun , Yang Yu , Zhuo Chen , Shizhu
Li a, Liping Duan a, d, *
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a National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO
Collaborating Centre for Malaria, Schistosomiasis, and Filariasis, Key laboratory of Parasitology
and Vector Biology of the Chinese Ministry of Health, Shanghai 200025, China
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b ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou 310058, China
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c Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of
Science and Technology, Shanghai 200237, China
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d Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
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*Corresponding author. Address: National Institute of Parasitic Diseases, Chinese Center for
Disease Control and Prevention, 207 Ruijin Er Road, Huangpu District, Shanghai 200025, China.
Tel.: +86-21-64377008. E-mail: duanlp1@chinacdc.cn
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Abstract
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Malaria and schistosomiasis are two of the most socioeconomically devastating parasitic
diseases in tropical and subtropical countries. Since current chemotherapeutic options are limited
and defective, there is an urgent need to develop novel antiplasmodials and antischistosomals.
Hemozoin is a disposal product formed from the hemoglobin digestion by some blood-feeding
parasites. Hemozoin formation is an essential process for the parasites to detoxify free heme, which
is a reliable therapeutic target for identifying novel antiparasitic agents. A series of novel carbazole
aminoalcohols were designed and synthesized as potential antiplasmodial and antischistosomal
agents, and several compounds showed potent in vitro activities against Plasmodium falciparum
3D7 and Dd2 strains and adult and juvenile Schistosoma japonicum. Investigations on the dual
antiparasitic mechanisms showed the correlation between inhibitory activity of β-hematin formation
and antiparasitic activity. Inhibiting hemozoin formation was identified as one of the mechanisms of
action of carbazole aminoalcohols. Compound 7 displayed potent antiplasmodial (Pf3D7 IC50 =
0.248 ꢀM, PfDd2 IC50 = 0.091 ꢀM) and antischistosomal activities (100% mortality of adult and
juvenile schistosomes at 5 and 10 ꢀg/mL, respectively) and exhibited low cytotoxicity (CC50 =
7.931 ꢀM), which could be considered as a promising lead for further investigation. Stoichiometry
determination and molecular docking studies were also performed to explain the mode of action of
compound 7.
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Keywords: carbazole aminoalcohols, Plasmodium falciparum, Schistosoma japonicum,
antiplasmodials, antischistosomals, hematin.
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1. Introduction
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Parasitic diseases represent a major global health problem. In 2010, the global
disability-adjusted life years (DALYs) of parasitic diseases are estimated to be over 102 000, which
are greater than some well-known diseases, such as rheumatic heart disease and diabetes (Murray et
al., 2012). However, these parasitic diseases are usually “neglected”, since they are prevalent
mostly in low-income developing countries and poor or marginalized communities. Malaria and
schistosomiasis are two typical representatives of these diseases. Most of their current
chemotherapeutic strategies suffer from serious deficiencies such as poor efficacy, unacceptable
toxicity, high costs and resistance occurrence. Therefore, novel and effective drugs are sorely
needed.
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Malaria, caused by infection with protozoan of the genus Plasmodium, is the most
socioeconomically devastating parasitic disease world widely, causing an estimated one million
deaths annually (WHO, 2013). Pregnant women and children under five years old are the most
affected populations. Plasmodium falciparum is the most virulent human malaria parasite, which is
responsible for the vast majority of the malaria-related deaths (Nosten et al., 2004). In the absence
of effective vaccines, chemotherapy is the important pillar for malaria treatment. However, the
global emergence of resistance strains results in the gradual loss of effectiveness of the marketed
drugs, including chloroquine, mefloquine and artemisinins (Dondorp et al., 2009).
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Schistosomiasis is a chronic and debilitating parasitic disease caused by blood-flukes of the
genus Schistosoma. It is the second major parasitic disease in the world after malaria with more
than 230 million individuals infected (Gryseels et al., 2006). Schistosoma japonicum is the most
infectious among the four human pathogenic Schistosoma species. (Jia et al., 2007).
Schistosomiasis japonica is especially prevalent in lake and marshland regions in Asia, where it still
remains significant health concern and considerable economic burden (Garjito et al., 2008; Zhou et
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al., 2012). Current chemotherapy relies on the only drug, praziquantel, which has been widely used
as an effective antischistosomal for decades. Praziquantel is very potent against the adult worms,
but much less effective against the juvenile worms (schistosomula) (Fenwick and Webster, 2006).
In addition, the adverse effect of mass treatment and long-term medication of praziquantel has
revealed available evidence for the emergence of praziquantel resistance in schistosomes (Melman
et al., 2009; Pica-Mattoccia et al., 2009). Given the lack of alternative chemotherapeutics, there is a
pressing need for new chemical entities for schistosomiasis treatment.
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It is known that antiplasmodials have been demonstrated to be able to kill schistosomas in vitro
and/or in vivo, such as artemisinins (Utzinger et al., 2007), mefloquine (Keiser et al., 2010),
chloroquine (Oliveira et al., 2004) and pyronaridine (Xue et al., 2013). The detail mechanisms of
these drugs exhibiting dual anti-parasitic activity are still unclear. One of the molecular mechanisms
involves hemozoin formation (de Villiers and Egan, 2009). Hemozoin is an aggregate of hematin
(oxidized heme) produced upon hemoglobin digestion by hematophagous organisms. It is the main
mechanism of heme detoxification in several blood-feeding organisms, including Plasmodium
(Noland et al., 2003), Schistosoma (Oliveira et al., 2000), Haemoproteus columbae (Chen et al.,
2001) and Rhodnius prolixus (Stiebler et al., 2010). Free heme (ferriprotoporphyrin IX) is toxic to
the parasites, because it can peroxidate lipids, produce oxygen radicals, inhibit enzyme activities
and damage cell membranes (Aft and Mueller, 1983; Aft and Mueller, 1984). Hence, how to
dispose free heme is of central importance in the physiological processes of hematophagous
organisms. To detoxify the free heme, the malaria parasites convert it into insoluble crystals, known
as hemozoin. A similar process is observed in schistosomes, and hemozoins are produced and filled
in the gut of the worms (Homewood et al., 1972). Since hemozoin formation is essential for the
survival of these parasites, inhibiting hematin aggregation represents an attractive drug target.
Indeed, plenty of evidence has indicated that antiplasmodial drugs with proved hemozoin formation
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inhibitory activity were effective for schistosomiasis, e.g. chloroquine (Oliveira et al., 2004),
mefloquine (Xiao et al., 2014), and pyronaridine (Auparakkitanon et al., 2006).
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In our previous work, a phenotypic in vitro screening against adult S. japonicum was
performed. Among the positive test results, two hits, JFD03612SC and BTB12253SC (Maybridge
database, Fig. 1), arose our interest. Both compounds have a carbazole aminoalcohol scaffold, and
caused 100% mortality of adult worms at 10 ꢀg/mL. Besides, further assay results indicated that
JFD03612SC exhibited moderate antiplasmodial activity against P. falciparum 3D7 strain (IC50 =
2.671 ꢀM, BTB12253SC was not tested). Carbazole occurs in a wide-range of biologically active
compounds, including antivirals (Yamada et al., 2012), antibiotics (Hurley et al., 2015),
antiplasmodials (Molette et al., 2013). In addition, the aminoalcohol functional group was
considered as a privileged structure for antischistosomal activity (Keiser et al., 2009). Thus, we
believe that the two hits are good starting points for discovering novel antiparasitic agents against P.
falciparum and S. japonicum.
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In this work, sixteen carbazole aminoalcohol derivatives were synthesized to ascertain the
importance of the carbazole core, the amine type and the stereochemical structure. Their
antiplasmodial activities against Pf3D7 and PfDd2 strains and antischistosomal activities against
adult and juvenile S. japonicum were determined. Additionally, β-hematin formation inhibitory
activities of target compounds have also been evaluated. Preliminary structure-activity relationships
(SARs) were discussed. Stoichiometry determination and molecular docking studies were carried
out, which helped to explain the mode of action of carbazole aminoalcohols.
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2. Materials and methods
2.1. General procedures for the synthesis of carbazole aminoalcohols
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Reagents and solvents were all purchased from Sigma-Aldrich, and generally were used
without further treatment. Melting points were determined in a B-540 Büchi apparatus. NMR
spectra were run on a Bruker AM-400 400 MHz spectrometer. Chemical shifts were given in ppm
(δ, TMS) and coupling constants in Hz. High resolution mass spectra (HRMS) were recorded on a
Thermo Q Exactive Orbitrap LC-MS/MS. Thin layer chromatography (TLC) was performed on
silica gel F254 plates from Merck. All yields were unoptimized and generally represented the result
of a single experiment.
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Synthesis of carbazole aminoalcohols was performed as previously described (Wang et al.,
2016). To a solution of 9-(oxiran-2-ylmethyl)-9H-carbazole (2a-c, 2 mmol) in EtOH (20 mL),
corresponding amines (6 mmol) were added. For amines with lower reactivity (e.g. arylamines),
BiCl3 (1 mmol) was also added. The reaction mixtures were heated to reflux for 6 h. The progress of
the reactions was monitored by TLC. The mixtures were quenched with water (10 mL) and
extracted with EtOAc (3×10 mL). The organic phases were washed with water (3×20 mL) and brine
(3×20 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The obtained
residues were purified by recrystallization from ethanol to afford target compounds. The
characterization data of compounds 6-8 and 12-16 were reported in our previous work (Wang et al.,
2016), and the characterization data of compounds 3-5, 9-11, (R)-7 and (S)-7) were given as
follows.
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2.1.1. 1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(pyrrolidin-1-yl)propan-2-ol (3). White solid (59%,
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over two steps), mp: 135.1-137.4℃. HRMS: m/z = 363.1023 [M+H]+. H NMR (400 MHz,
DMSO-d6) δ 8.30 (d, J = 2.0 Hz, 2H, Ar-H), 7.66 (d, J = 8.8 Hz, 2H, Ar-H), 7.48 (dd, J = 8.8, 2.1
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Hz, 2H, Ar-H), 4.96 (d, J = 4.2 Hz, 1H, OH), 4.78-4.26 (m, 2H, CH2), 4.04-3.97 (m, 1H, CH),
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2.56-2.36 (m, 6H, 3CH2), 1.73-1.60 (m, 4H, 2CH2). C NMR (100 MHz, DMSO-d6) δ 139.77,
125.94, 123.35, 122.37, 120.07, 111.83, 68.32, 59.47, 54.13, 47.58, 23.14.
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2.1.2. 1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(piperidin-1-yl)propan-2-ol (4). White solid (66%, over
two steps), mp: 140.3-141.3℃. HRMS: m/z = 377.1176 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ
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8.32 (d, J = 2.0 Hz, 2H, Ar-H), 7.67 (d, J = 8.8 Hz, 2H, Ar-H), 7.48 (dd, J = 8.8, 2.0 Hz, 2H, Ar-H),
4.94 (s, 1H, OH), 4.48-4.36 (m, 2H, CH2), 4.05-3.98 (m, 1H, CH), 2.45-2.24 (m, 6H, 3CH2),
1.55-1.34 (m, 6H, 3CH2). 13C NMR (100 MHz, DMSO-d6) δ139.73, 125.92, 123.34, 122.38,
120.12, 111.83, 68.77, 59.77, 54.76, 47.82, 25.61, 23.92.
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2.1.3. 1-(3,6-Dichloro-9H-carbazol-9-yl)-3-morpholinopropan-2-ol (5). White solid (51%, over two
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steps), mp: 136.3-137.4℃. HRMS: m/z = 379.0975 [M+H]+. H NMR (400 MHz, DMSO-d6) δ
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8.30 (s, 2H, Ar-H), 7.67 (d, J = 8.8 Hz, 2H, Ar-H), 7.48 (dd, J = 8.7 Hz, 2.1 Hz, 2H, Ar-H), 5.00 (d,
J = 5.0 Hz, 1H, OH), 4.53-4.21 (m, 2H, CH2), 4.05 (m, 1H, CH), 3.59-3.53 (m, 4H, 2CH2),
2.45-2.25 (m, 6H, 3CH2). 13C NMR (100 MHz, DMSO-d6) δ 139.70, 125.94, 123.36, 122.38,
120.09, 111.83, 66.72, 66.13, 62.10, 54.01, 47.72.
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2.1.4. 1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(hexylamino)propan-2-ol (9). White solid (43%, over
two steps), mp: 105.1-107.9℃. HRMS: m/z = 393.1496 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ
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8.31 (d, J = 2.0 Hz, 2H, Ar-H), 7.67 (d, J = 8.8 Hz, 2H, Ar-H), 7.47 (dd, J = 8.8, 2.1 Hz, 2H, Ar-H),
5.04 (s, 1H, OH), 4.48-4.19 (m, 2H, CH2), 3.95-3.93 (m, 1H, CH), 2.57-2.45 (m, 4H, 2CH2),
1.41-1.25 (m, 8H, 4CH2), 0.86 (t, J = 6.8 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 139.61,
125.92, 123.37, 122.34, 120.10, 111.68, 68.83, 53.13, 49.54, 47.33, 31.28, 29.62, 26.54, 22.11,
13.91.
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2.1.5. 1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(heptylamino)propan-2-ol (10). White solid (63%, over
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two steps), mp: 74.5-78.1℃. HRMS: m/z = 407.1645 [M+H]+. H NMR (400 MHz, DMSO-d6) δ
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8.31 (d, J = 2.0 Hz, 2H, Ar-H), 7.67 (d, J = 8.8 Hz, 2H, Ar-H), 7.47 (dd, J = 8.8, 2.1 Hz, 2H, Ar-H),
5.03 (s, 1H, OH), 4.48-4.21 (m, 2H, CH2), 3.95-3.92 (m, 1H, CH), 2.57-2.44 (m, 4H, 2CH2),
1.40-1.25 (m, 10H, 5CH2), 0.86 (t, J = 6.7 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 139.62,
125.93, 123.37, 122.34, 120.12, 111.71, 68.83, 53.13, 49.53, 47.34, 31.29, 29.65, 28.70, 26.82,
22.08, 13.93.
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2.1.6. 1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(octylamino)propan-2-ol (11). White solid (70%, over
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two steps), mp: 87.8-91.2℃. HRMS: m/z = 421.1811 [M+H]+. H NMR (400 MHz, DMSO-d6) δ
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8.29 (d, J = 2.0 Hz, 2H, Ar-H), 7.66 (d, J = 8.8 Hz, 2H, Ar-H), 7.45 (dd, J = 8.8, 2.0 Hz, 2H, Ar-H),
4.47-4.23 (m, 2H, CH2), 3.97-3.92 (m, 1H, CH), 2.57-2.43 (m, 4H, 2CH2), 1.38-1.22 (m, 12H,
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6CH2), 0.84 (t, J = 6.8 Hz, 3H, CH3). C NMR (100 MHz, DMSO-d6) δ 139.61, 125.91, 123.38,
122.35, 120.10, 111.67, 68.82, 53.11, 49.53, 47.32, 31.29, 29.65, 29.01, 28.74, 26.87, 22.10, 13.92.
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2.1.7. (R)-1-(butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol ((R)-7). White solid (68%,
over two steps), mp: 73.2-75.4℃. HRMS: m/z = 365.1181 [M+H]+. 1H NMR (400 MHz, DMSO-d6)
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δ 8.31 (d, J = 2.1 Hz, 2H, Ar-H), 7.70 (d, J = 8.8 Hz, 2H, Ar-H), 7.48 (dd, J = 8.8, 2.1 Hz, 2H,
Ar-H), 4.75-4.61 (m, 1H, OH), 4.48-4.29 (m, 2H, CH2), 4.05-3.98 (m, 1H, CH), 2.72-2.51 (m, 4H,
2CH2), 1.48-1.29 (m, 4H, 2CH2), 0.88 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ
139.63, 125.96, 123.37, 122.33, 120.12, 111.74, 68.75, 53.08, 49.12, 47.34, 31.71, 19.93, 13.92.
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2.1.8. (S)-1-(butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol ((S)-7). White solid (60%,
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over two steps), mp: 121.5-125.3℃. HRMS: m/z = 365.1179 [M+H]+. H NMR (400 MHz,
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DMSO-d6) δ 8.31 (d, J = 2.1 Hz, 2H, Ar-H), 7.68 (d, J = 8.8 Hz, 2H, Ar-H), 7.47 (dd, J = 8.8, 2.1
Hz, 2H, Ar-H), 4.72-4.63 (m, 1H, OH), 4.48-4.26 (m, 2H, CH2), 3.98-3.93 (m, 1H, CH), 2.59-2.47
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(m, 4H, 2CH2), 1.43-1.27 (m, 4H, 2CH2), 0.88 (t, J = 6.9 Hz, 4H, CH3). C NMR (100 MHz,
DMSO-d6) δ 139.61, 125.98, 123.41, 122.36, 120.14, 111.73, 68.26, 52.57, 48.76, 47.27, 31.02,
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2.2. In vitro P. falciparum whole cell assay
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Pf3D7 (chloroquine-sensitive) and PfDd2 (chloroquine-resistant) strains were used in an in
vitro blood stage culture to evaluate the antiplasmodial efficacy of carbazole aminoalcohols. The
strain cultures were prepared following the protocols described by Xu et al (Xu et al., 2013).
Intraerythrocytic parasites were synchronised to a 95% ring stage population using 5% sorbitol
solution. Chloroquine was dissolved in water (milli-Q grade) to prepare stock solution, and
carbazole aminoalcohols and dihydroartemisinin in DMSO. All the stock solutions were diluted
with 1640 incomplete medium to reach the corresponding dilutions. Synchronous ring-stage
parasites (1% parasitaemia and 2% haematocrit) were incubated in 96-well plates with serial
dilutions of test compounds or controls for 72 h at 37 °C. In all cases except chloroquine, the
highest final concentration of DMSO was 0.2%, which was found to be nontoxic to the parasites.
The antiplasmodial effect of carbazole aminoalcohols was determined by a SYBR Green I
fluorometric assay (Xu et al., 2013). IC50 values were determined using a growth/sigmoidal option
of Origin 8.0.
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2.3. In vitro assay for drug effect on adult and juvenile S. japonicum
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Adult schistosomes were harvested by dissection from mesenteric veins and livers of infected
mice, 34-38 days post-infection. Through perfusion with ice cold Hanks’ balanced salt solution
(HBSS) containing heparin, schistosomes were collected and rinsed with HBSS three times before
incubation. The in vitro culture mediums containing RPMI 1640 (with 10% calf serum), 100 IU/mL
streptomycin, 100 IU/mL penicillin sodium, and 0.25 g/mL amphotericin B were prepared to
maintain the schistosomes. Four pairs of schistosomes (four of both sexes) were placed in each well.
The plates were incubated at 37 °C in 5% CO2 for 2 h. Then test compounds (final drug
concentrations: 10 and 5 ꢀg/mL) were added. The final volume of each well was 4.0 mL. DMSO
was used as negative control. Phenotypes of the schistosomes, including motility, viability and
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morphological alterations, were monitored at 24, 48 and 72 h post-incubation. Worm death was
defined as no any motor activity observed in suckers and worm bodies for 2 min. No cultured
schistosomes were dead in control samples after 72 h incubation.
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S. japonicum cercariae were mechanically transformed to schistosomula, and stored in culture
medium (RPMI 1640 medium supplemented with 5% fetal bovine serum, 100 IU/mL penicillin and
100 ꢀg/mL streptomycin) at 37 °C in 5% CO2, as described by Keiser (Keiser, 2010). For drug
assay, schistosomula (50/well) were incubated with test compounds (final drug concentration: 10
ꢀg/mL) in a 40-well culture plate at 37°C in a 5 % CO2 incubator for 72 h. DMSO was used as
negative control. Assays were performed in duplicate. The activity status, survival time, mortality
and body morphology of the schistosomula were evaluated microscopically at 12 h, 24 h, 48 h and
72 h post-incubation.
2.4. Inhibition assay of β-hematin formation
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The inhibition assay of β-hematin was performed using the NP-40 detergent-mediated method
(Sandlin et al., 2011). Under acidic experimental conditions, hematin was allowed to form
β-hematin. DMSO solution (10 ꢀL) of test compounds at various concentrations was delivered to a
96-well plate, and then 20 ꢀL of NP-40 (30.55 mM) and 70 ꢀL of deionised H2O were added into
each well. The hematin stock solution (25 mM) was prepared by dissolving hematin in DMSO
through sonicating, then 178 ꢀL of which was suspended in a 2 M acetate buffer (pH 4.8). For each
well, 100 ꢀL of the homogenous suspension was added to reach 0.5 M final buffer and 100 mM
hematin. DMSO was used as negative control. Amodiaquine (100 ꢀM, final concentration) was
used as a positive control. The plates were covered and incubated at 37 °C for 4 h. Analysis was
conducted by using the pyridine-ferrichrome method. A solution of 50% (v/v) pyridine, 20% (v/v)
acetone, 30% (v/v) H2O, and 0.2 M HEPES buffer (pH 7.4) was prepared, and 32 ꢀL of this was
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delivered to each well to reach a final pyridine concentration of 5% (v/v). In order to give assistance
to hematin dispersion, additional acetone (60 ꢀL) was also added. The UV-vis absorbance was
recorded by a Bio-Rad Model 680 XR microplate reader at 405 nm. The IC50s of β-hematin
formation were determined using GraphPad Prism software. Assays were performed in triplicate,
and repeated twice.
232
2.5. Cytotoxicity assay on WI38 cells
233
234
235
236
237
238
239
240
241
WI38 cells were grown and harvested at log phase. Cells were plated in a 96-well plate at
10,000 cells per well in 180 ꢀL of dulbecco's modified eagle medium (DMEM) or minimal essential
medium (MEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. After
12 h incubation at 37 °C in 5% CO2 to allow the cells to adhere, 20 ꢀL of 2-fold serial dilutions of
compounds were added in the well in triplicate. The final concentrations of compounds were 50, 25,
12.5, 6.25, 3.13 and 1.57 ꢀM. Plates were incubated for another 2 days at 37°C in 5% CO2. Then
supernatants were removed, and 90 ꢀL of fresh medium and 10 ꢀL of MTT solution were added.
After 4 h incubation, the supernatants were removed again and 110 ꢀL of DMSO was added in each
well. The plates were swirled gently for 10 min, and then read the absorbance at 490 nm.
242
2.6. Drug-hematin interaction assay
243
244
245
246
247
248
249
250
Stoichiometry determination by the continuous variation method (Job’s plot) was carried out to
study the drug-hematin interaction through determining the spectral changes. The aqueous DMSO
(40%, v/v) solution of 10 M hematin and test compounds were prepared as previously described by
Auparakkitanon et al (Auparakkitanon et al., 2003). The combined concentration of drug and
hematin was kept constant (10 ꢀM). For each test drug, 11 solutions of drug and hematin
combinations in different molar ratios were prepared as follows: 0:10, 1:9, 1:4, 3:7, 2:3, 1:1, 3:2,
7:3, 4:1, 9:1, and 10:0. Spectra between 240 and 700 nm were read on a Beckman Coulter DU730
spectrophotometer.
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2.7. Molecular docking
252
253
254
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Docking simulation was performed by using CDOCKER module (Discovery Studio, version
2.1, Accelrys). The three-dimensional structure of heme was obtained from the free heme crystal
structure (PDB: 3P5Q). The Fe atom was charged +3, and the Fe (III) protoporphyrin IX form was
used for docking simulation. Hemozoin formation occurs within the plasmodium digestive food
vacuoles (pH 4.8). At relevant acidic conditions, both N atoms of compound 7 are protonated
(Wang et al., 2016). Accordingly, they were charged +1 for docking simulations. For each isomer
of 7, random conformations were generated by utilizing CHARMm field molecular dynamics (1000
steps), and then docked into the defined binding site with a radius set as 11 Å. Other parameters
were set as default. The binding conformations of (R)-7 and (S)-7 with heme were determined and
ranked according to the calculated CDOCKING energy. Among the top 30 docking poses, the most
stable binding modes were showed in Fig. 4. Visualization of docking results was performed with
DS Viewer Lite from Accelrys.
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265
3. Results and discussion
3.1. Synthesis of carbazole aminoalcohols
266
267
268
269
The synthetic routes of carbazole aminoalcohol derivatives are summarized in Fig. 2. Reaction
of carbazoles (1a-c) and epichlorohydrin (racemic or enantiomerically pure) in the presence of
KOH afforded the epoxypropane intermediates (2a-c), which subsequently reacted with appropriate
amines to obtain corresponding target compounds 3-16.
270
3.2. Antiplasmodial activity
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272
273
The in vitro antiplasmodial activities of carbazole aminoalcohols were determined against
chloroquine-sensitive Pf3D7 and chloroquine-resistant PfDd2 strains. Chloroquine and
dihydroartemisinin were used as the positive controls, and the results are summarized in Table 1.
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In general, most of the carbazole aminoalcohols exhibited moderate to potent antiplasmodial
activities against Pf3D7 and PfDd2 strains. Retaining the dichloronated carbazole core,
manipulating the amine tail of 3 altered the activity. All the compounds with alkylamine tails (6-11)
displayed remarkable antiplasmodial activity with IC50s in the submicromolar range against Pf3D7
strain and nanomolar range against PfDd2 strain. Among them, n-hexylamino (9, Pf3D7 IC50 =
0.274 ꢀM, PfDd2 IC50 = 0.047 ꢀM) and n-octylamino groups (11, Pf3D7 IC50 = 0.132 ꢀM, PfDd2
IC50 = 0.054 ꢀM) were the preferred substituents. For the compounds with aniline substituents
(12-14), a drop of potency was observed. On the other hand, dihalogenated carbazole was found to
be a privileged core structure after varying the substituents of carbazole. Comparing with compound
7 (Pf3D7 IC50 = 0.248 ꢀM, PfDd2 IC50 = 0.091 ꢀM), eliminating the chlorine atoms of carbazole
core (15, Pf3D7 IC50 = 0.760 ꢀM, PfDd2 IC50 = 0.334 ꢀM) resulted in 3-fold decrease in potency in
both strains. In addition, in order to find out whether the stereochemistry of the secondary hydroxyl
group exerted influence on potency, the R- and S- enantiomers of 7 were prepared and evaluated.
Both enantiomers showed similar antiplasmodial activity ((R)-7, Pf3D7 IC50 = 0.344 ꢀM, PfDd2
IC50 = 0.172 ꢀM; (S)-7, Pf3D7 IC50 = 0.378 ꢀM, PfDd2 IC50 = 0.112 ꢀM).
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Table 1. The in vitro antiplasmodial, antischistosomal and β-hematin formation inhibitory activities of carbazole aminoalcohols
Adult S. japonicum
Juvenile S. japonicum
β-Hematin
Pf3D7
PfDd2
worm-killinga
worm-killinga
Compd.
X
R
IC50 (ꢀM)
IC50 (ꢀM)
10 ꢀg/mL
5 ꢀg/mL
10 ꢀg/mL
NT
% Inhibitionb
IC50 (ꢀM)
Cl
Cl
Cl
Cl
Cl
Cl
＋
＋
－
＋
＋
＋
－
－
NT
±
2.671±0.870
NT
NT
NT
10.76
NT
-
3 (JFD03612SC)
NT
-
4 (BTB12253SC)
NT
1.294±0.329
0.379±0.063
0.248±0.032
0.292±0.044
NT
0
-
5
6
7
8
＋
0.089±0.015
0.091±0.026
0.121±0.041
55.97
82.31
48.06
151.503±10.454
91.839±7.732
143.820±6.470
＋
±
＋
NT
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Cl
Cl
Cl
Cl
＋
＋
＋
－
±
±
＋
＋
0.274±0.063
0.179±0.044
0.132±0.059
5.404±0.793
0.047±0.010
0.054±0.010
0.054±0.006
NT
81.85
74.74
50.59
0
54.983±5.872
91.646±4.667
65.377±4.899
-
9
10
11
12
±
＋
NT
NT
Cl
Cl
－
－
NT
NT
NT
NT
4.283±0.684
5.950±0.230
NT
NT
0
0
-
-
13
14
H
Br
Cl
Cl
-
＋
＋
＋
＋
NT
－
－
＋
＋
NT
NT
NT
＋
0.760±0.022
0.492±0.100
0.344±0.054
0.378±0.091
0.015±0.008
0.334±0.067
0.059±0.012
0.172±0.032
0.112±0.030
0.231±0.024
0
-
15
16
61.51
75.13
80.09
97.8
117.430±8.767
118.892±7.136
105.517±8.416
61.011±5.553
(R)-7
(S)-7
＋
-
NT
Chloroquine
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-
-
-
-
-
-
NT
NT
＋
NT
NT
＋
NT
NT
－
NT
NT
-
0.005±0.0002
NT
100
-
NT
Dihydroartemisinin
Amodiaquine
NT
NT
15.893±1.288
-
Praziquantel
291 NT: not tested.
a
292
＋: All the cultured S. japonicum were dead; －: none of the cultured S. japonicum was dead; ±: half of the cultured S. japonicum were dead.
293 b % Inhibition assay was performed at a concentration of 100 ꢀM.
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3.3. Antischistosomal activity
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All prepared target compounds were further evaluated for their antischistosomal activity
against adult and juvenile S. japonicum. The results were summarized in Table 1.
297
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300
301
302
303
304
305
306
307
308
309
310
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312
Sixteen compounds were tested, and twelve of them killed all adult S. japonicum at 10 ꢀg/mL
after 72 h exposure. Among them, seven compounds killed worms with 50-100% mortality at 5
ꢀg/mL. In consistent with the tendency observed in antiplasmodial activity, most of the compounds
with alkylamine tails (6-11) showed significant antischistosomal activity, and the most potent
compound 7, with an n-butylamino group, killed adult S. japonicum with 100% mortality at 5
ꢀg/mL. Both enantiomers of 7, (R)-7 and (S)-7, were as effective as the racemate. In accordance
with the results of P. falciparum whole cell assay, compounds with arylamine substituents (12-14)
suffered a significant loss of potency. In addition, compared with the dichlorinated carbazole
derivative 7, the dibromo- (15) and non- substituted (16) derivatives exhibited reduced activity
against adult worms. Based on the adult worm killing ability, representative compounds were
further evaluated for their antischistosomal activity against schistosomula (Table 1). All the tested
compounds (6, 7, 9-11, (R)-7 and (S)-7) demonstrated significant juvenile worm killing ability,
causing 100% mortality at 5 ꢀg/mL in 24 h. The WHO recommended activity criterion of hit
compounds for schistosomiasis is 100% inhibition of motility of schistosoma adults at 5 ꢀg/mL
(Nwaka and Hudson, 2006). Based on this criterion, compound 7 can be considered for further in
vivo animal studies.
313
3.4. β-Hematin formation inhibitory activity
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According to the above results, most of the carbazole aminoalcohols showed not only
antiplasmodial but also antischistosomal activities, especially those with alkylamine substituents.
Plasmodium and schistosome are both hematophagous organisms, and the hemozoin formation is
crucial for the survival of these parasites. We speculated that inhibiting hemozoin formation was
one of the mechanisms of action of these compounds, similar to the known β-hematin formation
inhibitors with dual antiparasitic activities (e.g. chloroquine, mefloquine and pyronaridine). In order
to verify our hypothesis, the inhibition assay of β-hematin (synthetic hemozoin) formation was
performed. The known inhibitors, chloroquine and amodiaquine, were used as positive controls.
322
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327
328
329
330
331
332
As shown in Table 1, in the preliminary assay, ten compounds showed measurable inhibition
ratios at the concentration of 100 ꢀM. The presence of an alkylamine tail (6-11) was favorable to
potency. Compounds with alicyclic amine groups (3 and 5) or arylamine groups (12-14) were not
able to inhibit β-hematin formation. The removal of two halogens on the carbazole moiety (15)
resulted in a dramatic decrease in potency, which demonstrated the essential role of
dihalogen-substituted carbazole core. Further assay results indicated that almost all the compounds
possessing alkylamine groups (6-11) exhibited significant β-hematin formation inhibitory activity.
Especially, compounds with n-butylamino (7, IC50 = 91.839 ꢀM), n-hexylamino (9, IC50 = 54.983
ꢀM), n-heptylamino (10, IC50 = 91.646 ꢀM), and n-octylamino (11, IC50 = 65.377 ꢀM) groups
displayed potent activity equivalent to that of chloroquine (IC50 = 61.01 ꢀM). The stereochemistry
of the linker had no influence on potency.
333
334
The antiplasmodial activity of carbazole aminoalcohols have been identified in several
phenotypic screenings, but their mode of action remains unclear. Recently, it has been reported that
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Plasmodium falciparum Hsp90 was a plausible target of carbazole aminoalcohols (Wang et al.,
2016), while there is still no clue of their mechanism of action on schistosomes. In this work, three
kinds of biological assay revealed a similar SAR pattern: (1) dichlorinated carbazole acted as a
privileged core; (2) an alkylamine tail was beneficial for activity; conversely, arylamine substituents
imparted negative effect to potency; and (3) stereochemistry of the secondary hydroxyl group had
no influence on potency. The SARs indicated that β-hematin formation inhibitory activity of target
compounds showed correlation with their antiplasmodial and antischistosomal activities, especially
the latter. Although further investigations are needed, the current data basically confirmed our
hypothesis that inhibiting hemozoin formation was one of the mechanisms of action of carbazole
aminoalcohols.
345
3.5. Cytotoxicity
346
347
348
349
350
351
352
353
354
355
In order to assess the toxicity of carbazole aminoalcohols, representative compounds (6-11)
were tested for their cytotoxicity against human embryonic lung fibroblast WI38. The results were
summarized in Table 2 (CC50s). Generally, all of the tested compounds showed moderate
cytotoxicity with CC50s in the micromolar range. The selectivity indices (SI), the ratio of
cytotoxicity (CC50) and antiplasmodial activity (IC50 for Pf strains), were also calculated (Table 2),
allowing to identify the potential therapeutic windows. All the compounds showed acceptable SI
values in chloroquine-sensitive Pf3D7 strain ranging from 19 to 32, and more satisfactory SI were
observed in chloroquine-resistant PfDd2 strain ranging from 45 to 126 (Table 1). Particularly,
compound 7 was the safest molecule with a CC50 value of 7.931 ꢀM, a SI [CC50/IC50 (Pf3D7)] value
of 32, and a SI [CC50/IC50 (PfDd2)] value of 87.
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Table 2. The in vitro cytotoxicity and selectivity indices of carbazole aminoalcohols
SIb
Compd. CC50 (ꢀM)a
CC50/IC50 (Pf3D7) CC50/IC50 (PfDd2)
7.268
7.931
6.259
5.920
5.141
3.773
7.686
9.343
19
32
21
22
28
29
22
24
82
87
52
126
95
70
45
83
6
7
8
9
10
11
(R)-7
(S)-7
357
358
359
a 50% Cytotoxic concentration, WI38 cell line, means of two independent experiments.
b Selectivity Index (SI) was calculated as CC50/IC50 ratio.
3.6. Stoichiometry determination by the continuous variation method (Job’s plot)
360
361
362
363
364
365
The binding of carbazole aminoalcohols with hematin was investigated by the continuous
variation technique (Job’s plot). A sharp peak at 401 nm was observed in hematin solution at pH 7.4,
indicating that monomeric hematin predominated under the experimental conditions. The addition
of represented carbazole aminoalcohols (6-11) led to the decrease of absorption in the Soret band,
indicating the association of compounds and hematin. When the molar ratios of hematin and
compounds were 1:1, changes in absorbance intensity reached the maximum (see Fig. 3 for
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368
369
representative plot of 7). It demonstrated the formation of a 1:1 drug:hematin complex. In contrast
with 7 and 9, compounds 6 and 8 produced minor changes in absorbance (data not shown),
suggesting that they interacted weakly with hematin, which was in agreement with their less potent
β-hematin formation inhibitory activity.
370
3.7. Molecular docking studies
371
372
373
Molecular docking studies were performed to predict the binding modes of representative
compound 7 (R and S isomers) with heme by utilizing the C-DOCKER program within Discovery
Studio 2.1 software package.
374
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377
378
379
380
381
382
383
384
385
As expected, the docking models (Fig. 4) indicated that (R)-7 and (S)-7 exhibited similar
binding modes with heme. Both isomers could form stable non-covalent complexes with heme,
which consequently led to the inhibition of hemozoin formation. As observed from the axial view
(Fig. 4a and 4b), the carbazole core formed a π-π stacking interaction with porphyrin ring. It
probably played a key role in the stability of drug-heme complex. The carbazole core was not right
above the iron center, but located toward one side of porphyrin ring, presumably to form preferable
π-π interaction. Similar binding modes were observed in amodiaquine-, chloroquine-, and quinine-
heme complexes (Leed et al., 2002; de Dios et al., 2004). The aminoalcohol functional groups (NH
and OH) of (R)-7 and (S)-7 formed three hydrogen bonds with both carboxyls of heme, respectively,
which probably further stabilized the complex. The side view (Fig. 4c and 4d) showed that the
distances between carbazole centroid and heme porphyrin planar of (R)-7 and (S)-7 were 3.78 and
3.73Å, respectively, which satisfied the optimal interplanar distance value of approximate 3.0-4.0 Å
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(de Sousa et al., 2015).
4. Conclusions
388
389
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393
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395
396
In this work, novel carbazole aminoalcohols were designed and prepared, and their
antiplasmodial and antischistosomal potential have been confirmed. Most of the compounds
displayed significant β-hematin formation inhibitory ability, which showed correlation with their
dual antiparasitic activities, identifying that inhibiting hemozoin formation was one of their
mechanisms of action. The preliminary SARs confirmed the importance of the amine type and the
dichlorinated carbazole core. Job’s plot revealed that carbazole aminoalcohol compound interacted
with hematin through forming a 1:1 complex. It is noteworthy that compound 7 showed not only
potent dual antiparasitic activities but also low cytotoxicity, which could be developed as a
promising lead compound for further investigation.
397
Acknowledgments
398
399
400
401
402
This work was supported by China Postdoctoral Science Foundation funded project (grant
number 2015M571147), the National Natural Science Foundation of China (grant numbers
21502181 and 21302054), Shanghai Municipal Commission of Health and Family Planning funded
project (grant number 201440537) and the project of echinococcosis prevention and control - Ganzi
workstation.
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