Journal Pre-Proof: European Journal of Medicinal Chemistry

Journal Pre-proof
A comprehensive review on β-lapachone: Mechanisms, structural modifications, and

therapeutic potentials
Qijie Gong, Jiabao Hu, Pengfei Wang, Xiang Li, Xiaojin Zhang
PII: S0223-5234(20)30934-X
DOI: https://doi.org/10.1016/j.ejmech.2020.112962
Reference: EJMECH 112962
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 August 2020

Revised Date: 3 October 2020
Accepted Date: 19 October 2020
Please cite this article as: Q. Gong, J. Hu, P. Wang, X. Li, X. Zhang, A comprehensive review on β-
lapachone: Mechanisms, structural modifications, and therapeutic potentials, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112962.
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A comprehensive review on β-lapachone:
Mechanisms, structural modifications, and
therapeutic potentials
Qijie Gong a, Jiabao Hu a, Pengfei Wang b, Xiang Li b,*, and Xiaojin Zhang a,*
a
Jiangsu Key Laboratory of Drug Design and Optimization, and Department of Chemistry, China
Pharmaceutical University, Nanjing 211198, China
b
Department of Pharmaceutical Engineering, China Pharmaceutical University, Nanjing 211198,
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China
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Corresponding authors: E-mail addresses: zxj@cpu.edu.cn (X. Zhang), lixiang@cpu.edu.cn (X. Li)
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Abstract: β-lapachone (β-lap, 1), an ortho-naphthoquinone natural product isolated from the lapacho
tree (Tabebuia avellanedae) in many regions of South America, has received extensive attention due to
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various pharmacological activities, such as antitumor, anti-Trypanosoma cruzi, anti-Mycobacterium

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tuberculosis, antibacterial, and antimalarial activities. Related mechanisms of β-lap have been widely
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investigated for a full understanding of its therapeutic potentials. Numerous derivatives of β-lap have
been reported with aims to generate new chemical entities, improve the corresponding biological
potency, and overcome disadvantages of its physical and chemical properties and safety profiles. This
review will give insight into the pharmacological mechanisms of β-lap and provide a comprehensive
understanding of its structural modifications with regard to different therapeutic potentials. The
available clinical trials related to β-lap and its derivatives are also summarized.
Keywords: β-lapachone; natural product; quinone derivatives; structural modification
1
Abbreviations
ACC, Acetyl-CoA carboxylase; AEs, Adverse events; AMPK, Adenosine 5’-monophosphate-activated
protein kinase; Anti-T. cruzi, Anti-Trypanosoma cruzi; ATP, Adenosine triphosphate; CBR1, Carbonyl
reductase 1; CPR, NADPH-cytochrome P450 reductase; Cys, L-Cysteine; DLT, Dose limited toxicity;
DNA, Deoxyribonucleic acid; DUBs, Deubiquitinases; H2O2, Hydrogen peroxide; HP-β-CD,
hydroxypropyl-β-cyclodextrin; Hsp90, Heat shock protein 90; hTERT, Human telomerase reverse
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transcriptase; HUVECs, Human umbilical vein endothelial cells; IC50, Drug concentration that leads to
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lysis of 50 % of the parasites; IDO1, Indoleamine 2,3-dioxygenase 1; mRNA, messenger RNA; LC50,
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Drug concentration that leads to damage of 50 % of the cells; 5-LO, 5-Lipoxygenase; MIC: Minimal
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inhibitory concentration; MTD, Maximum tolerated dose; NAD(P)H, Nicotinamide adenine
dinucleotide phosphate; NAD, Nicotinamide adenine dinucleotide; NQO1, NAD(P)H:quinone

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oxidoreductase-1; PARP-1, poly(ADP-ribose)polymerase-1; PKA, Protein kinase A; PPARα,

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Peroxisome proliferator-activated receptor α; ROS, Reactive oxygen species; RTKs, Receptor tyrosine
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kinases; SI, Selectivity index: Corresponds to the ratio between the LC50 and IC50; Sirt1, Silent mating
type information regulation 2 homolog 1; SR, Selectivity ratio; SOD, Superoxide dismutase; T. cruzi,
Trypanosoma cruzi; T/C, Tumor proliferation rate; TGI, Tumor growth inhibition; USP2, Ubiquitin
specific protease 2; VEGFR2, Vascular endothelial growth factor receptor 2.
2
1. Introduction
Natural products play critical roles in drug development and serve as vital sources of the new drug
discovery [1, 2]. It has been suggested that approximately 35 percent of drugs on the market were
derived from natural products, of which about 25 percent were originated from natural plants [3].
Although researchers could discover novel lead compounds from small-molecule compound libraries
via high-throughput screening technology [4], natural products still retain their unique advantages and
are irreplaceable in the process of new drug discovery, especially in the therapeutic areas of antitumor
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agents and antibiotics [5].
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β-Lapachone (β-lap, 1, Fig. 1), 3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b] pyran-5,6-dione, is an
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ortho-naphthoquinone natural product isolated from the lapacho tree (Tabebuia avellanedae) in many
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regions of South America [6, 7]. Over the past decades, numerous studies have revealed that β-lap
exhibited a variety of pharmacological effects, such as antitumor [8-10], anti-Trypanosoma cruzi

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(anti-T. cruzi) [11, 12], anti-Mycobacterium tuberculosis [13, 14], antibacterial [15, 16], antimalarial
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[17, 18], anti-metabolic syndrome [19], and anti-inflammatory [20] activities. It worth noting that β-lap
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acts as anti-infective agents toward various pathogens, including Trypanosoma cruzi, Plasmodium
falciparum, and Mycobacterium tuberculosis. Trypanosoma cruzi (T. cruzi) is the causative agent of
Chagas disease infecting millions of people in Latin America, which can be divided into three forms
(epimastigote, amastigote, and trypomastigote forms) according to its life cycle [21]. Plasmodium
falciparum is the etiologic agent of the malignant form of malaria [17]. Mycobacterium tuberculosis is
the cause of tuberculosis and still represents the major threat of global death, responsible for millions of
deaths annually worldwide [22]. Considering that cancers and infectious diseases remain major threats
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to human health [23, 24], it is of therapeutic importance to develop corresponding new drugs, for
instance, based on β-lap with regard to its diverse bioactivity.
Given that β-lap exerts broad biological activities, related mechanisms of β-lap are complex and
remain to be fully elucidated [25]. Among them, the antitumor mechanism for β-lap has been
well-investigated; the antitumor activity raised by β-lap was associated with the bioreductive activation
by NAD(P)H: quinone oxidoreductase-1 (NQO1) that led to rapid production of reactive oxygen
species (ROS) and multiple regulations on the downstream pathways related to cancer growth and
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development [26-36]. It has also been suggested that the antitumor effects of β-lap were related to the
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inhibition of topoisomerase I [37], topoisomerase II [38], indoleamine 2,3-dioxygenase 1 (IDO1) [39],
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telomerase [40], and heat shock protein 90 (Hsp90) [41]. Notably, β-lap has completed multiple phase I
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and phase II clinical trials for cancer treatments in the form of ARQ 501 and ARQ 761 [42-45].
However, several disadvantages of β-lap, such as poor water solubility and narrow therapeutic
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windows, limited its clinical applications [46, 47]. Concerning the broad and significant therapeutic
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potentials of β-lap, numerous derivatives have been reported with aims to generate new chemical
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entities, improve the corresponding biological potency, and overcome its disadvantages in
physicochemical properties and safety profiles [17, 22, 48-50].
Previously, Green et al. summarized related patents on the antitumor activity of β-lap in the past
two decades [51]. da Silva Júnior group reviewed the synthetic methods and biological activity for
some important derivatives of β-lap [52, 53]. This review will give insight into the pharmacological
mechanisms of β-lap and provide a comprehensive understanding of its structural modifications with
regard to different therapeutic potentials. The available clinical trials related to β-lap and its derivatives
are also summarized.
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2. Pharmacological mechanisms of β-lap
Given that β-lap has shown various pharmacological activities and significant therapeutic potentials,
great efforts have been devoted to investigating its corresponding mechanisms. The pharmacological
mechanisms of β-lap are complex and its various biological activities remain to be fully elucidated [25].
As shown in Fig. 1, the antitumor mechanism of β-lap was mainly associated with the redox cycling
activated by NQO1-mediated bioreduction, which led to a rapid generation of ROS and thus induced a
series of downstream biological events related to the suppression of tumor cell growth [26-35]. Besides,
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the antitumor effects of β-lap were also found to be related to the inhibition of a panel of enzymes, such
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as topoisomerase I [37], topoisomerase II [38], IDO1 [39], telomerase [40], and Hsp90 [41]. As for the
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mechanisms of β-lap beyond antitumor activities, such as antibacterial and trypanocidal effects [54],
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were also suggested to be ascribed to the rapid ROS generation triggered by NQO1 that could cause
antibacterial and antitrypanosomal effects [55-57]. Moreover, the changes in the ratio between NAD+
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and NADH mediated by NQO1 bioreduction were found to be associated with the therapeutic effects of
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β-lap on metabolic syndrome [58].

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Fig. 1. Pharmacological mechanisms of β-lap.
2.1 Antitumor mechanisms
2.1.1 β-Lap and NQO1
NQO1 is a flavoenzyme that catalyzes the two-electron reduction of quinone substrates; it can convert
β-lap into hydro-β-lap (2) [49]. The reduction product 2 is chemically unstable and can spontaneously
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turn back into β-lap in the presence of oxygen in cells [32], resulting in the futile redox cycling of β-lap
[59]. It was reported that such futile redox cycle of β-lap could lead to the activation of protein kinase
A (PKA), inducing caspase activation-mediated apoptosis [60]. Furthermore, in this redox cycling, it
consumes a large amount of the reductive cofactor, nicotinamide adenine dinucleotide phosphate
(NAD(P)H) during the reduction of quinone β-lap to dihydroquinone 2, and produces a large amount of
ROS such as superoxide anion (O2•–) during the oxidation of 2 back into β-lap by cellular O2. The
formed superoxide anion could be converted to hydrogen peroxide (H2O2) by superoxide dismutase
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(SOD) in the body. The resulting ROS, O2•– and H2O2, could both cause DNA cleavage [32]. In
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addition, enhanced ROS can trigger downstream signaling cascades and induce apoptosis in cancer
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cells [61-65]. Thus, β-lap can induce cancer cell apoptosis by triggering excessive ROS formation
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through the NQO1-meidated redox cycling.
It was suggested that ROS-mediated apoptosis raised by β-lap was mainly related to the
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poly(ADP-ribose)polymerase-1 (PARP-1) signal pathway [61, 62]. PARP-1 is an enzyme that plays a
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significant role in DNA repair. The ROS, generated by the NQO1-mediated futile redox cycling,
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induces DNA damage and thus causes the PARP-1 hyperactivation [61]. The continuous
hyperactivation of PARP-1 could lead to intensive NAD+/ATP depletion and thus suppress the DNA
repair, resulting in cell apoptosis and cell death[62].
Recently, β-lap was found to be an inhibitor of ubiquitin specific protease 2 (USP2) as a
consequence of ROS formation raised by β-lap in cancer cells [63]. USP2 as a type of deubiquitinases
(DUBs), belongs to a subclass of cysteine proteases. It was reported that the overexpression of USP2
could protect tumor cells from apoptosis, mainly through blocking the proteasomal degradation of
related proteins [64]. The silence of USP2 was found to facilitate apoptosis in several human tumor
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cells [66]. Recently, studies have revealed that the key Cys residues in the catalytic sites of DUBs, such
as USP2, could be oxidized in the presence of high levels of ROS [67-70]. Hence, the enhanced ROS
level caused by the redox cycling of β-lap inhibited and inactivated USP2 by oxidizing the thiol moiety
into cystine sulfinic or sulfonic acids (Fig. 1) [63, 64]. Upon the deactivation of USP2, the proteasomal
degradation pathways were initiated in tumor cells, being responsible for the antitumor activity of
β-lap.
The calcium-mediated signaling pathway was also found to be associated with the ROS-mediated
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cancer cell death for β-lap [65]. The reduction of β-lap by NQO1, using NAD(P)H as a reductive
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cofactor, caused a dramatic and rapid decrease in the cellular levels of NAD(P)H, leading to the
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activation of the calcium ion channel and the consequent release of calcium from the endoplasmic
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reticulum [71, 72]. The rapidly increased levels of calcium in cytoplasmic triggered the action of
pumping calcium out of the cytosol to maintain normal levels of calcium; it consumed a mass of ATP
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and caused depolarization of the mitochondrial membrane [72]. Such continuous depolarization of
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mitochondrial could interrupt the intracellular ion homeostasis, allowing the extracellular calcium to
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significantly enter the cell. The significant increase in the intracellular calcium levels induced the
activation of the protease and endonuclease, ultimately resulting in cancer cell apoptosis [73-76].
Recently, Rodrigues et al. reported 5-lipoxygenase (5-LO) as a target of β-lap by machine learning
approach study [77]. It was suggested that β-lap was converted into hydro-β-lap 2 through
NQO1-mediated bioreduction; the latter inhibited 5-LO and caused cancer cell death.
2.1.2 β-Lap and topoisomerase
β-Lap could cause apoptosis by inhibiting the activity of DNA topoisomerase I and Ⅱ [37, 78]. These
enzymes catalyze the breaking and binding of DNA strands in DNA replication, transcription, and
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repair [79]. Li et al. reported that β-lap was an inhibitor of topoisomerase I, and that it blocked the
DNA repair in cancer cells upon the radiation exposure from X-rays [37, 80]. Interestingly, the pattern
that β-lap bound to topoisomerase Ⅱ was different from that of camptothecin, a well-recognized
inhibitor of topoisomerase I. It was reported that Camptochecin bound to the complex of DNA and
topoisomerase I instead of DNA or topoisomerase I [37]. In contrast, β-lap did not bind to the complex
consisting of DNA and topoisomerase I; it only bound to the free topoisomerase I protein to form
complex. Subsequently, the complex interacted with DNA to form an inactive ternary complex [37],
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which was incapable of releasing free topoisomerase I, leading to insufficient DNA repair and
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enhancement of DNA damage [37]. Additionally, Liu et al. reported that the cytotoxicity of β-lap
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toward yeast strains was related to the deletion of the topoisomerase I gene and was independent of the
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topoisomerase I expression [78]. Further research revealed that β-lap could inactivate topoisomerase Ⅱ
in the yeast system through the reversible formation of the topoisomerase II-cleavable complexes, as a
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consequence, leading to DNA damage and cell apoptosis [78].

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2.1.3 β-Lap and IDO1

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The apoptosis induced by β-lap was also found to be related to the inhibition of the IDO1 enzyme [81].
IDO1, a rate-limiting enzyme that catalyzes the degradation of L-tryptophan [39], acts as an immune
protector for tumor cells, which does not directly participate in the growth of tumor cells [82, 83].
Recently, Muller et al. reported that β-lap was a potent inhibitor of IDO1 in a non-competitive manner
[81]. Thus, β-lap was able to suppress the IDO1-mediated immune protection for tumor cells. Besides,
the inhibition of IDO1 by β-lap could block the metabolic pathway of L-tryptophan and inhibit the
biosynthesis of nicotinamide adenine dinucleotide (NAD), significantly aggravating the depletion of
the NAD stores in cancer cells [84, 85]. Immunoinhibition along with the deletion of NAD raised by
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β-lap synergistically led to the growth inhibition of cancer cells.
2.1.4 β-Lap and telomerase
Telomerase is an enzyme that regulates the activity of telomeres. Telomeres are important units in
biological genetic processes and stabilizes the chromosome ends of eukaryotic cells to avoid losing the
genetic information, being responsible for the unlimited proliferation in most of the tumor cells [86-89].
Choi et al. reported that β-lap suppressed the activity of telomerase in a dose-dependent manner by
downregulating the mRNA level of the human telomerase reverse transcriptase (hTERT) [25, 40],
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leading to cancer cell death.
2.1.5 β-Lap and Hsp90 -p

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Hsp90 was found to be highly expressed in the NQO1-overexpressing tumor cells and human umbilical
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vein endothelial cells (HUVECs) [41]. It was reported that β-lap induced the cleavage of Hsp90 upon
the enhanced oxidative stress levels in tumor cells and led to the degradation of the Hsp90-associated
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oncoproteins and key receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor
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receptor 2 (VEGFR2), resulting in suppression of tumor angiogenesis [41]. Thus, the antitumor effects
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raised by β-lap was also related to the Hsp90 pathway.
2.2 Mechanisms for the other biological activities
In addition to antitumor activity, β-lap presented a range of other biological activities, such as
anti-T. cruzi [11, 12], antibacterial [15, 16], and anti-metabolic syndrome [19] activities. The related
mechanisms beyond the antitumor activity for β-lap were suggested to be in relation to the production
of ROS and the exhaustion of NAD(P)H initiated by the NQO1-mediated redox cycling (Fig. 1) [55, 90,
91]. It was reported that β-lap could accept a single electron to form a semiquinone radical, which
tended to oxidize spontaneously [55, 90]. During the autoxidation of semiquinones, oxygen underwent
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univalent reduction and produced O2- [92]. The formed O2- was prone to be oxidized to generate H2O2
spontaneously or by the action of the superoxide dismutase. Moreover, O2- and H2O2 could convert
nonenzymatically to hydroxyl radical [92]. These products of the partial reduction of oxygen could
trigger free-radical reactions, which caused the peroxidation of polyunsaturated fatty acids of
membrane lipids and damaged the DNA [55, 90], which were responsible for the trypanocidal and
antibacterial activities of β-lap. In addition, NAD+ and NADH served as the fundamental mediators of
energy metabolism; the enhanced NAD+ level may ultimately cause the effects of calorie restriction in
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mammals [58]. Recently, it was reported that β-lap could enhance the NADH oxidation in the
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NQO1-sufficient cells through the NQO1-mediated futile redox cycling [58]. β-Lap-induced NADH
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oxidation contributed to the phosphorylation and activation of adenosine 5’-monophosphate-activated
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protein kinase (AMPK), a key molecule in the regulation of energy metabolism. In addition, enhanced
NADH oxidation could also lead to the potent inhibition toward the phosphorylation of acetyl-CoA
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carboxylase (ACC), which played a crucial role in fatty acid synthesis. Both the inhibition of ACC and
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activation of AMPK could inhibit lipogenesis and provoke fatty acid oxidation. Imbalance of the
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NAD+/NADH ratio caused by β-lap could also stimulate silent mating type information regulation 2
homolog 1 (Sirt1), an NAD+ dependent deacetylase [91]. The activation of Sirt1 could lead to the
upregulation of peroxisome proliferator-activated receptor α (PPARα), which was responsible for the
transcription of genes encoding enzymes related to the fatty acid oxidation [93]. Thus, β-lap exhibited
the anti-metabolic syndrome effect by inhibiting lipogenesis and enhancing fatty acid oxidation (Fig.
1).
3. Structural modifications on β-lap
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β-Lap has attracted extensive attention concerning its diverse and important pharmacological activities,
including antitumor, anti-Trypanosoma cruzi, anti-Mycobacterium tuberculosis, antibacterial, and
antimalarial activities (Fig. 2). Structurally, β-lap consists of a benzene ring, an ortho-quinone ring, and
a dihydropyran ring, namely A-, B-, and C-rings (Fig. 2), respectively. In this review, we will discuss
the structural modification of β-lap based on the classification of A-, B-, and C-ring modified
derivatives with their corresponding biological activities.
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Fig. 2. Chemical structure of β-lap and its therapeutic potentials.
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3.1 Antitumor derivatives

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3.1.1 C-ring modified derivatives

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3.1.1.1 Dihydropyran derivatives

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With a quite similar structure to β-lap, rhinacanthone (3, Fig. 3), also an ortho-quinone natural
product, was found to be cytotoxic against a panel of tumor cells. The structural differences between
β-lap and 3 lie in their gem-dimethyl groups installed on the different sites of C-ring, suggesting that
the gem-dimethyl substitution in β-lap was not essential for its antitumor activity and it may be
substituted by other groups. In light of this finding, Kongkathip et al. synthesized several analogs of
β-lap by introducing methyl or hydroxyl groups on the dihydropyran C-ring [94]. Among them, 4 and 5
exerted the potent activities against HeLa, HepG2, and KB cancer cells with IC50 values ranging from
0.92 to 3.22 μM, while 6 with hydroxyl moiety showed lower inhibitory activities toward these cancer
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cells with IC50 values ranging from 7.61 to 24.13 μM (Fig. 3). Rocha et al. reported that 7 (Fig. 3) with
an iodine group introduced on the C-ring, showed potent cytotoxic activity toward thirteen tumor cell
lines (IC50 = 0.02-2.5 μM) [95]. Notably, the cytotoxicity of 7 was 22 to 35-fold more potent compared
to that of β-lap toward AGP-01, ACP-02, and HCT-29 cancer cell lines. Furthermore, 7 was found to be
much more selective to tumor cells than β-lap and doxorubicin, a well-recognized anticancer drug. For
instance, in assays with the lineage HL-60 (human promyelocytic leukemia) using PBMC (blood
mononuclear cells) as control, the SI (selectivity index) values for β-lap, 7, and doxorubicin were 827,
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1690 and 520, respectively [95]. Camara et al. introduced hydroxymethyl or acetoxymethyl groups on
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the C3 position of the C-ring of β-lap and evaluated their antitumor activities against BGMK, HEP-2,
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NCI-H292, and MCF-7 cancer cell lines [96]. For instance, compounds 8 and 9 (Fig. 3) showed potent
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inhibitory activity toward MCF-7 cells with IC50 values of 5.51 and 2.73 μM, respectively [96].
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Fig. 3. C-ring modified dihydropyran derivatives of β-lap with antitumor activities.
da Silva Júnior et al. firstly introduced an azide moiety to the C4 site of C-ring (10) and then
generated a novel series of the triazole-containing derivatives of β-lap utilizing click chemistry (Fig. 3).
These triazole derivatives, as represented by 11-16 (Fig. 3), were further evaluated their cytotoxicity
against six cancer cell lines, including PC-3, HCT-116, HL-60, MDAMB-435, and SF-295 cancer cells
[97, 98]. All the compounds showed potent inhibitory activity against HL-60 cells with IC50 values
ranging from 0.66 to 1.88 μM, except for 16 (IC50 > 8.4 μM), which were comparable to β-lap (IC50 =
1.65 μM). In the case of MDAMB-435 cells, all the triazole compounds also exerted potent
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cytotoxicity (IC50 < 2 μM). Among these compounds, 15 was the most active with IC50 values ranging
from 0.41 to 1.59 μM toward the six tumor cell lines; however, their selectivity to tumor cells (SI =
0.8-3) still needed to be improved [98].
da Silva Júnior et al. synthesized a series of selenium- or sulfur-containing derivatives modified at
the C3 site of C-ring and evaluated their antitumor activities against ten cancer cell lines [99, 100]. It
was suggested that the introduced selenium or sulfur atoms could serve as a second redox center in
addition to the ortho-quinone moiety. In HL-60 cancer cells, 17-24 (Fig. 3) showed potent activity with
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IC50 values ranging from 0.53 to 1.8 μM except 20 [99]. In MOLT-4 cancer cells, 17, 19, 22, and 24
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were found to be the most active compounds (IC50 of 0.73-1.89 μM) [99]. For HCT-116 cells, 20
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exhibited the most potent activity with an IC50 of 0.95 μM [100]. Interestingly, 19, with a bromo
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substituent at the para position of the phenylselanyl moiety, showed broad and potent cytotoxicity
against all the tested cancer cells with IC50 values ranging from 0.94 to 1.72 μM. These results
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suggested that the introduction of a second redox center, such as a selenium or a sulfur atom, did
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contribute to the antitumor activity for the ortho-quinone derivatives of β-lap.

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Further, da Silva Júnior et al. designed and synthesized eight derivatives by a pharmacophore
hybridization strategy to introduce both the triazole and chalcogens at C-ring and evaluated their
cytotoxic effects against PC-3, NCI-H460, SF-295, HCT-116, and HL-60 cell lines [9]. Most of them
presented potent growth inhibitory activities against all tested tumor cells. Among them, 25 (Fig. 3)
was found to be the most active against L-929, HCT-116, HL-60, PC-3, NCI-H460, and SF-295 cancer
cells with IC50 values of 0.36, 0.37, 0.59, 1.06, 1.32, and 1.48 μM, respectively. These results indicated
that the introduction of a second Se-containing redox center and a triazole moiety provided a new
chemical type of β-lap derivatives with promising antitumor activity.
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3.1.1.2 Dihydrofuran derivatives
Dihydrofuran derivatives of β-lap were designed by reducing the C-ring from the six-membered
dihydropyran ring to a dihydrofuran ring. As shown in Fig. 4, compound 26, an ortho-quinone natural
product named nor-β-lapachone (nor-β-lap), could be regarded as a derivative of β-lap with a reduced
C-ring; it exhibited potent inhibitory activities against a panel of cancer cells, such as MDAMB-435,
HCT-8, SF-295, and HL-60 cells with IC50 values of 0.31, 1.36, 1.58, and 1.75 μM, respectively [101].
This evidence suggested that changing of the dihydropyran C-ring into dihydrofuran would affect little
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to its antitumor activity for β-lap.
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By changing the gem-dimethyl groups at the C2 site of C-ring to mono-substituted groups, da
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Silva Júnior et al. reported 27 and28 and evaluated their antitumor activities against four human
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leukemia cell lines including Jurkat, MOLT-4, K562, and HL-60 cells [102]. Importantly, 27 showed an
IC50 value of 1.38 μM toward Jurkat cells, while 28 had an IC50 value of 1.11 μM toward HL-60 cells,
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which was comparable to that of 26 (IC50 = 1.34 μM for Jurkat cells, IC50 = 1.86 μM for HL-60 cells).
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Based on the structure of 27, da Silva Júnior et al. and Costa et al. further introduced substituted
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triazoles to the C2 position of dihydrofuran ring [98, 103]. As represented by 29, it significantly
inhibited the cell growth toward a panel of cancer cells, such as HL-60 (IC50 = 1.15 μM), MDAMB-435
(IC50 = 1.17 μM), PC-3 (IC50 = 1.29 μM), SF-295 (IC50 = 1.43 μM), and HCT-116 (IC50 = 1.68 μM) [98].
In addition, 30 with triazole moiety directly attached the C2 site, showed good toxicity against Caco-2
cancer cells (IC50 = 4.48 μM), while it was found to be much less toxic to normal Vero cells (LC50 = 28
μM), showing a moderate SI of 6.25 between cancer and normal cells [103].
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Fig. 4. C-ring modified dihydrofuran derivatives of β-lap with antitumor activities.
In addition to the C2 substituted derivatives, the C3 position of the dihydrofuran ring also served
as a potential modification site. Camara et al. installed a hydroxymethyl or acetoxymethyl on the C3
site of C-ring to obtain 31 and 32 (Fig. 4), which were highly active against MCF-7 cancer cells with
IC50 values of 0.46 and 0.6 μM, respectively [96]. Moreover, Goulart group synthesized a series of
C3-site substituted triazole derivatives 33-38 (Fig. 4) and evaluated their cytotoxic effects against six
cancer cell lines [101]. Among them, 37 was the most active compound toward all the tested cancer cell
lines with IC50 values ranging from 0.43 to 1.83 μM. In MDAMB-435 cancer cells, 37 (IC50 = 0.43 μM)
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was even more potent than the positive drug doxorubicin (IC50 = 0.86 μM); importantly, 37 showed 2-
to 4-fold less active to normal L-929 cell. In addition, 36 and 38, with aromatic or alicyclic substituents
on the triazole moiety, both showed significant cytotoxicity toward all the tested cancer cells with IC50
values below 5 μM. Compound 39 (Fig. 4), with alkyl substituent on the triazole moiety, reported by
Silva group, was found to be highly active against leukemia cell lines, such as CEM (IC50 = 0.34 μM)
and MOLT (IC50 = 0.05 μM) cells, which was comparable to doxorubicin (IC50 = 0.15 μM for CEM
cells, IC50 = 0.07 μM for MOLT cells) [104].
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Encouraged by the good performance of 36 against HL-60 (IC50 = 2.1 μM) [101], da Silva Júnior
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group further introduced various electron-withdrawing or electron-donating groups onto the para
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position of phenyl of 36, as represented by 40-42 (Fig. 4), showing potent growth inhibition toward all
lP
the tested leukemia cell lines, inducing Jurkat, MOLT-4, K562, and HL-60 cells [102]. Among them, 42
was the most cytotoxic against the four cancer cells with IC50 values in the range of 0.74-1.99 μM.
na
Another type of the C-ring modified triazole derivatives, as represented by 43 and 44 (Fig. 4), were
ur
reported by da Silva Júnior et al. to possess moderate cytotoxicity toward five tested cancer cells
Jo
including PC-3, HCT-116, HL-60, MDAMB-435, and SF-295, with IC50 values in the range of
1.34-3.35 μM [98].
Dihydrofuran quinone derivatives with arylamino substituted at the C3 site of C-ring, such as
45-51 (Fig. 5) were obtained by Pinto and Ferreira group [102, 105, 106]. Most of these compounds
presented great cytotoxicity toward six tested cancer cells with IC50 values below 2 μM [105]. It was
worth noting that 46 (IC50 = 0.19 μM) and 51 (IC50 = 0.39 μM) were more potent than doxorubicin
(IC50 = 0.88 μM) in MDAMB-435 cells [106]. Most of them showed similar toxicity to normal cells
with poor selectivity between cancer and normal cells. Interestingly, 48 and 51 with an
18
ortho-substituent on phenyl showed a higher selectivity to tumor cells with SI values between
5.76-87.8 [106]. In addition, Ferreira et al. reported a series of pyrazole substituted derivatives
modified at the C3 site of C-ring. [107]. All of them were certified to possess potent activity against
OVCAR-8, SF-295, HCT-116, and HL-60 cancer cells with IC50 values ranging from 0.16 to 2.13 μM.
Especially, 52 (Fig. 5) was found to be the most active compound against OVCAR-8, SF-295,
HCT-116, and HL-60 with IC50 values of 0.99, 1.05, 0.72, and 0.34 μM, respectively.
of
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-p
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lP
na
ur
Jo
19
Fig. 5. C-ring modified dihydrofuran derivatives of β-lap with antitumor activities.
In addition to the arylamino substituted derivatives, Pinto group reported a series of alkyloxy
substituted derivatives modified at the C3 site of C-ring [106]. These alkoxy derivatives, as represented
by 53-55 (Fig. 5), showed good cytotoxicity against SF-295, HCT-8, and HL-60 cancer cells with IC50
values in the range of 1.04-5.93 μM. While in the case of MDAMB-435 cells, 53-55 were highly active
with IC50 values in the range of 0.27 and 0.59 μM, being slightly more efficacious than doxorubicin
(IC50 = 0.88 μM). Importantly, 53-55 had much higher SI values as 10.05, 10.59, and 17.22,
of
ro
respectively, when compared to doxorubicin (SI = 0.48) [106]. Further, Ferreira et al. synthesized
-p
several novel glycosyl derivatives 56-58 (Fig. 5), which were highly active toward OVCAR-8, SF-295,
re
HCT-116, and HL-60 cancer cells with IC50 values between 0.16-0.89 μM [107]. The introduction of
lP
glycosyl may contribute to improving the drug-like properties, such as water solubility.
Using a molecular hybridization strategy, antitumor fragments such as para-naphthoquinone and

na
chalcone were introduced to the C3 site of 26 to obtain new chemical types of dihydrofuran derivative
ur
of β-lap [108]. For instance, 59 (Fig. 5) showed significant cytotoxicity toward a panel of cancer cells,
Jo
including SF-295, OVCAR-8, HCT-116, and HL-60 cells, with IC50 values in the range of 0.45-1.33
μM. However, it had poor selectivity between cancer cells and normal cells. Compound 60 (Fig. 5),
being highly sensitive to HL-60 cells with IC50 values in the range of 0.04-0.2 μM, showed a
significantly increased selectivity between cancer cells and normal cells (SI = 79) as compared with
that of doxorubicin (SI = 12), indicating that 60 was a promising chemical type for further research.
[108]
Being similar to the selenium-containing dihydropyran derivatives, da Silva Júnior et al. designed
several selenium-containing dihydrofuran derivatives by introducing a second redox center [100, 109].
20
Compounds 61-62 were found to be greatly potent toward the tested cancer cell lines, including HL-60,
HCT-116, PC-3, SF-295, MDA-MB-435, and OVCAR-8 cells, with IC50 values ranging from 0.07 to
0.38 μM [109]. Compounds 63-64 also possessed good cytotoxicity toward the tested cancer cells (IC50
= 1.06-2.52 μM) with some exceptions [100, 109]. Importantly, 61 (SI = 19.8) showed a higher
selectivity to HL-60 cells when compared to doxorubicin (SI = 9.1) [109]. In addition, Goulart group
used a similar strategy to design a series of 3-substituted thio-nor-β-lap with their cytotoxic activity
evaluated against HCT-116, K562, PC3, and SF-295 cell lines [110]. Most of the thionaphthoquinone
of
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derivatives showed moderate to potent activity against all the tested cells with IC50 values below 5 μM.
-p
However, some of them were found to be even more cytotoxic against normal cells than tumor cells.
re
Among them, 65 showed the lowest IC50 against K562 (IC50 = 0.9 μM). Importantly, it was observed
lP
that 65 showed considerable selectivity to HCT-116 and K562 cells with SI values of 2.96 and 2.11,
respectively [110]. These results emphasized the importance of selenium-containing pharmacophore in

na
developing novel quinone derivatives of β-lap with good cytotoxicity and selectivity.
ur
Recently, da Silva Júnior group reported several fluorescent probes based on 26 by introducing a
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fluorescent group at C-ring for imaging both in cells and in vivo [111-113]. For instance, 66 (Fig. 6),
with a benzothiadiazole moiety, showed good fluorescent cell imaging properties in MDA-MB-231
cancer cells. It exerted potent cytotoxic activities toward twenty cancer cell lines with IC50 values in the
range of 0.48 and 1.24 μM [111]. Additionally, probes 67-69 (Fig. 6) also retained potent antitumor
activities against a panel of tested cancer cells. With a fluorescent BODIPY moiety introduced, they
were suggested to be promising probes for imaging both in cells and in vivo and useful tools for
antitumor mechanistic investigations [112, 113].
21
of
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Fig. 6. C-ring modified antitumor derivatives of β-lap with a fluorescent group.
3.1.1.3 Tetrazolopyrimidine derivatives

-p
re
By merging a heterocycle, tetrazolo[1,5-a]pyrimidine, into the C-ring of β-lap, two novel series of
lP
naphthoquinone-fused tetrazolopyrimidines were designed and synthesized by Wu group [114, 115].

na
Among the derivatives in series A, 70 (Fig. 7) was found to be the most active compound with IC50
ur
values of 1.18 and 0.22 μM against HepG2 and HCT-116 cells, respectively, being approximately
Jo
20-fold more potent than Taxol [114]. The derivatives in series B featured a spiro scaffold as shown in
71 (Fig. 7). Among this series, 71 showed good cytotoxicity (IC50 = 2.86 μM) and selectivity (SI = 20.6)
toward HepG2 cancer cells [115].
Fig. 7. Tetrazolopyrimidine derivatives of β-lap with antitumor activity.
22
3.1.2 B-ring modified derivatives
Burton et al. functionalized the carbonyl group in B-ring of β-lap with an imine moiety leading to
novel arylimino derivatives and evaluated their antiproliferative activity against nine tumor cell lines
[116]. These arylimino derivatives, as represented by 72-73 (Fig. 8), showed comparable cytotoxicity
to β-lap with IC50 values ranging from 0.19 to 2.56 μM. The change in the redox cycling center did not
necessarily affect overall activity and selectivity of the parent quinone β-lap, suggesting that it was an
alternative approach in the modification of antitumor quinones such as β-lap. Further, Araújo et al.
of
ro
reported a B-ring modified oxime derivative 74 [117]. It presented high cytotoxicity against SF-295
-p
and HL-60 cancer cells with IC50 values of 3.47 and 3.84 μM, respectively. Moreover, 74 exhibited
re
6-fold selectivity to tumor cells over the normal fibroblast cells, being more selective toward cancer
lP
cells than its precursor β-lap. These results indicated that introducing the oxime group at B ring may be
a valuable way to improve safety profiles for the ortho-quinone antitumor agents, such as β-lap.
na
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23
Fig. 8. B-ring, A-ring, and C- and A-rings modified antitumor derivatives of β-lap.
3.1.3 A-ring modified derivatives
Hong group introduced various electron-withdrawing or electron-donating groups onto the A-ring
of β-lap and evaluated the cytotoxic effect against OCI-LY3 cell lines [118]. The structure-activity
relationships (SARs) suggested that the introduction of electron-withdrawing groups on the C8 position
of β-lap contributed to its antiproliferative activity, while the methoxy group on the C9 position of
β-lap was unfavorable for the activity. For instance, 75 (Fig. 8) with cyano presented the best
of
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cytotoxicity against OCI-LY3 cancer cells with an IC50 value of 1.5 μM, while 76 with a methoxy
-p
group showed a significant decrease in activity with an IC50 value of 17 μM. Furthermore, Padrón
re
group introduced hydroxyl or methoxyl substituents on the different positions of A-ring leading to eight
lP
derivatives and evaluated the antiproliferative activities against WiDr, SW1573, HeLa, and HBL-100
cancer cells [119]. Among them, 77 (Fig. 8) was found to be the most active toward all the tested
na
cancer cells with GI50 values ranging from 0.029 to 2.0 μM. The results suggested that the introduction
ur
of a hydroxyl group at the C7 site adjacent to the quinone redox center was beneficial for the antitumor
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activity of β-lap.
3.1.4 A- and C-ring modified derivatives
Ferreira group reported a series of A- and C-ring modified derivatives of β-lap by introducing
both a hydroxyl at the C7 site of A-ring and a substituted phenyl at the C2 site of C-ring and had them
biologically evaluated against several cancer cells, including SF-295, HCT-8, HL-60, and
MDAMB-435 cells [120]. All of them were found to be much more sensitive and selective to
MDAMB-435 cells with IC50 values in the range of 0.11-0.53 μM. Especially, 78 (Fig. 8), with an IC50
value of 0.11 μM, was shown to be the most active compound toward MDAMB-435 cells, being 8-fold
24
more potent than doxorubicin (IC50 = 0.88 μM). As compared with 78, the introduction of halogens to
the para of phenyl, as presented by 79-81 (Fig. 8) led to a slight decrease in activity toward
MDAMB-435 cells with IC50 values ranging from 0.15 to 0.21 μM), while it increased, in general, the
selectivity toward MDAMB-435 over other three tested tumor cells [120]. Besides, Moreira et al. also
introduced alkyl groups at the C2 site of C-ring to generate 82-83 and had them biologically evaluated
against several cancer cells, including HL-60, MCF-7, PC-3, HCT-116, and SNB-19 cells [121].
Compounds 82-83 exhibited high activities against all the tested cells with IC50 values in the range of
of
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0.42-3.06 μM. Notably, They were most sensitive to HL-60 cells with IC50 values ranging from 0.42 to
-p
0.56 μM and SI values ranging from 5.32 to 13.18 [121].
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3.1.5 Modifications focusing on the NQO1-targeting bioreductive activity and drug-like properties
lP
β-Lap has completed several phase I/II clinical trials for cancer treatment, however, several
disadvantages of β-lap limited its further application in clinical therapy [122]. Those disadvantages
na
mainly included: 1) β-lap is unstable in the small and large intestines because the pyran ring (C-ring) of
ur
β-lap was tended to be hydrolyzed to ring-opening products under an alkaline environment [123],
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which were toxic to normal cells and caused side effects [122]. 2) Cyclodextrin plays an important role
in improving the solubility of poorly soluble compounds [124]. However, a high proportion of
hydroxypropyl-β-cyclodextrin (HP-β-CD) was required for the formulation for intravenous
administration of β-lap due to its poor water solubility (0.043 mg/mL) [125], which probably caused
serious side effects such as hemolytic anemia [126]. 3) Besides the two-electron oxidoreductase NQO1,
β-lap could also be bioreductively activated by the one-electron oxidoreductases, which were widely
expressed in normal tissues such as the liver, thus leading to potential hepatotoxicity [127, 128]. All
these aspects mentioned above may contribute to the narrow therapeutic window of β-lap. Recently, for
25
overcoming the above shortcomings, Wang et al. reported a pH/ROS dual-responsive nanomedicine of
β-lap, which was proved to exhibited potent antitumor activity and low toxicity [129]. Furthermore,
structural modifications have been performed to solve these disadvantages. The following section will
mainly discuss the structural modifications of β-lap focusing on the NQO1-targeting bioreductive
activity and drug-like properties, such as physicochemical properties and safety profiles.
3.1.5.1 Modifications focusing on the NQO1-targeting bioreductive activity and C-ring stability
The antitumor effects of β-lap were mainly related to the bioreductive reduction mediated by
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NQO1. To obtain a preliminary SAR information on NQO1-targeting activity, our group set out to
-p
design and synthesize a series of the dihydrofuran derivatives of β-lap, as represented by 26, 28, and
re
84-88 (Fig. 9) [130]. These compounds were evaluated for their NQO1 reduction activity and
lP
cytotoxicity against A549 (NQO1+) and A549 cell lines pretreated with the NQO1 inhibitor dicoumarol
(A549+DIC/NQO1-). The results showed that, in general, these dihydrofuran derivatives with reduced
na
size of C-ring were all capable of being efficiently reduced by NQO1. It was suggested that the
ur
introduction of methyl and methoxy group on the C- and A-ring, respectively, was beneficial for the
Jo
antitumor activity. For instance, 84 (kcat/Km = 1.9 ± 0.4 × 106 M-1 s-1) with three methyl groups on the
C-ring presented higher catalyzed reduction rate than 26, 28 and 85 (kcat/Km = 0.8-1.4 × 106 M-1 s-1).
86-88 with methoxy group on the A-ring also exhibited a higher reduction rate than 84. Especially, 87
(kcat/Km = 3.1 ± 0.5 × 106 M-1 s-1) was more efficacious than β-lap (kcat/Km = 2.6 ± 0.5 × 106 M-1 s-1).
Besides, 87 showed a good inhibitory activity toward the NQO1-overexpressing A549 cancer cells with
an IC50 value of 6.8 μM, which was comparable to β-lap (IC50 = 7.0 μM), and it also showed an
increased selectivity ratio (SR) of 6.0 between A549 (NQO1+) and A549+DIC (NQO1-) cells when
compared with that of β-lap NQO1 (SR = 3.0). These results suggested that the C-ring and A-ring of
26
β-lap are well-tolerated for modification, retaining the NQO1 reduction acidity and NQO1-targeting
antitumor activity.
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na
ur
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Fig. 9. Modifications of β-lap focusing on the NQO1-targeting bioreductive activity and C-ring
stability. The unit for the NQO1 reduction rate is mmol NADPH/min/mmol NQO1.
Considering that the dihydropyran ring of β-lap was prone to be metabolically ring-opened, a new
aromatic furan-fused scaffold, as in 89 (Fig. 9), was initially designed by merging the structures of
another natural ortho-quinone Tanshinone IIA (TSA) with β-lap [122], which was considered to be
much more stable and not prone to be ring-opened in the physiological condition. The resulting
compound, 89, was able to be reduced by NQO1, but it showed a much lower reduction rate (765 ± 22
μmol NADPH/min/μmol NQO1) when compared with the lead compound β-lap (1146 ± 64 μmol
27
NADPH/min/μmol NQO1) (Fig. 9). Taking account that the binding site of NQO1 was an “L-shaped”
pocket, as illustrated in a crystal complex structure of NQO1 and its potent inhibitor dicoumarol (DIC)
(Fig.10, left) [131, 132], we set out to induce a side chain at the C2 site of C-ring of 89 based on
molecular docking and overlap analysis (Fig.10, right), making the derivatives as “L-shaped”
molecules to fully occupy the “L-shaped” pocket of NQO1. As expected, all of the L-shaped
derivatives showed significantly increased reduction rates by NQO1 compared with the planar
compound 89. Especially, those derivatives substituted with aromatic side chains, as represented by
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90-93 (Fig. 9), were found to be reduced by NQO1 highly efficiently, with NQO1 reduction rates of
-p
1213-1302 μmol NADPH/min/μmol NQO1, being even better than β-lap. In addition, 91-93 showed
re
good inhibitory activity toward A549 (NQO1+) cancer cells with IC50 values of 4.9, 6.0, and 4.7 μM,
lP
respectively, which were comparable to β-lap (IC50 = 5.7 μM). Notably, among them, the “L-shaped”
derivative 92 with the best NQO1 reduction activity was not sensitive to NQO1 deficient cancer cells
na
and showed a high selectivity between A549 (NQO1+) and H596 (NQO1-) cancer cells with SR>50,
ur
being more selective than β-lap (SR = 4.0) [122]. These results suggested that the L-shaped derivatives
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may fit well with the NQO1 binding pocket and show a specific NQO1-targeting antitumor activity,
proving a valuable modification strategy for reducing the potential off-target side effects of the quinone
substrates, such as β-lap.
28
Fig. 10. Left: Dicoumarol (pink) was in the crystal structure of 2F1O and the surface of them was
of
added [131, 132]; Right: Dicoumarol (pink) and compound 89 (blue) were in the crystal structure of
ro
2F1O and the surrounding amino acid residues were shown. Dashed lines represented hydrogen bonds
[122].
-p
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Recently, Liu group utilized another strategy to increase the stability of the C-ring [133]. They
lP
designed a new lactam-fused scaffold by replacing the pyran ring with a six-membered lactam because
na
the six-membered lactam moiety showed favorable stability during metabolism. In addition, being
ur
similar to the furan-fused scaffold derivatives, molecular docking studies showed that introducing
Jo
additional side chains to occupy a side binding pocket contributed to the binding affinity, as represented
by 94 (Fig. 9). Compound 94 was proved to be reduced by NQO1 with high efficiency; it showed an
NQO1 reduction rate of 2770 μmol NADPH/min/μmol NQO1, being significantly fast than β-lap (1220
μmol NADPH/min/μmol NQO1). More importantly, 94 exhibited good inhibitory activity toward A549,
HepG2, and MCF-7 cells with IC50 values in the range of 2.23-3.13 μM, comparable to β-lap (IC50 of
2.83-4.17 μM). In vivo evaluation showed that 94 (20 mg/kg) could effectively suppress tumor growth
by an inhibitory rate of 50.3% in the HepG2 xenografts mouse model with no obvious side-effects
observed. These results emphasized that the lactam-fused scaffold was beneficial for improving the
29
NQO1-targeting antitumor potency, which was worthy of further development.
3.1.5.2 Modifications focusing on the NQO1-targeting bioreductive activity and water solubility
Although 92 gained an improved NQO1-targeting activity, the poor water solubility remained unsolved.
The intrinsic water solubility of 92 (0.021 mg/mL) was even lower than β-lap (0.043 mg/mL), probably
due to the introduction of the hydrophobic phenyl group. Thus, HP-β-CD was still required in the
injection formulation, leading to potential side effects such as hemolytic anemia. Consequently, our
group continued the research to find new derivatives with improved water solubility and, designed and
of
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synthesized a series of oxazole derivatives by introducing additional nitrogen atoms [126]. The results
-p
showed that the oxazoles 95-97(Fig. 11) (1220-1522 μmol NADPH/min/μmol NQO1) possessed
re
comparable NQO1 reduction rates to its furan analog 92. The intrinsic solubility of 97 (0.044 mg/mL)
lP
was also slightly improved compared with 92 (0.021 mg/mL). Further introducing a piperazine moiety
to the side chain as in 98-99 significantly improved the water solubility. For instance, 98 showed an
na
intrinsic water solubility of 0.125 mg/mL, being approximately three times more soluble than β-lap
ur
(0.125 mg/mL), and an even better solubility at pH = 3 (0.5 mg/mL), thus avoiding the use of HP-β-CD
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in the injection formulation. The docking studies on 98 and NQO1 suggested that the nitrogen atom in
the oxazole ring could form a hydrogen-bonding interaction with the Tyr128 residue, and the
water-soluble side chain, benzylpiperazine, fit with the binding pocket formed by several aromatic
residues in the catalytic site of NQO1 (Fig. 12). In vitro studies showed that 98 had good cytotoxicity
toward A549（NQO1+）cancer cells with an IC50 value of 4.6 μM. Further, the antitumor efficacy of 98
was evaluated in vivo in the A549 tumor xenografts mouse model, showing an increased relative tumor
proliferation rate (T/C) of 41.4% at the dose of 30 mg/kg with iv administration compared with that of
β-lap (T/C = 29.5%). In addition, the weights of the mice treated by 98 during three-week treatments
30
were similar to that of the vehicle control group, emphasizing a preliminary safety profile of 98 [126].
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lP
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Fig. 11. Modifications of β-lap focusing on the NQO1-targeting bioreductive activity and water
ur
solubility
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Fig. 12. The docking pose of 98 in the active site of NQO1 (PDB ID: 2F1O). Black dot lines indicate
hydrogen-bonding interactions [126].
31
3.1.5.3 Modifications focusing on the NQO1/CPR selectivity and safety profiles
Considering that β-lap would also bioreductively activated by the one-electron oxidoreductases [128],
such as NADPH-cytochrome P450 reductase (CPR), overexpressed in human normal liver cells, we
proposed that the selectivity between the two-electron oxidoreductase NQO1 and one-electron
oxidoreductases, such as CPR, could cause potential hepatotoxicity and be responsible for the narrow
therapeutic window of β-lap [128]. Initially, we tested β-lap and its representative derivatives targeting
NQO1 and found that they all showed poor NQO1/CPR selectivity, as seen in 89 (SR (NQO1/CPR) =
of
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0.63), 92 (SR (NQO1/CPR) = 1.22), and β-lap (SR (NQO1/CPR) =1.36) (Fig.13) [128]. Consequently,
-p
we set out a structure-selectivity (NQO1/CPR) studies based on the naphthoquinone scaffold, and over
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twenty quinone analogs with diversity structures designed, synthesized, and evaluated their NQO1 and
lP
CPR reduction rates. The results showed that the presence of methyl groups on the A-ring had a great
impact on the NQO1/CPR selectivity. For instance, as in 100-103 (Fig.13), these methyl-substituted
na
compounds with a sustained NQO1 activity (905-1623 μmol NADPH/min/μmol NQO1) showed an
ur
improved NQO1/CPR selectivity (SR(NQO1/CPR) = 1.38-6.37) compared with that of 89

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(SR(NQO1/CPR) = 0.63). Especially, among them, 100 with C7/C8 dimethyl substituents, possessed a
highest SR (NQO1/CPR) value of 6.37). Further changing the position or number of the methyl groups
on A-ring and C-ring all led to the decrease in selectivity as in 101-103 (Fig.13). Docking studies
suggested that that the two carbonyl groups on the B-ring could interact with Tyr126 residue, and the
two adjacent methyl groups on the A-ring could form C-H...π interactions with Trp105 and Phe178
residues, respectively (Fig. 14A) [59].
32
O O
O O
O
O
O O
O R
β-lapachone (1) 89 92 R = p-F-PhNH
SR (NQO1/CPR) = 1.36 SR (NQO1/CPR) = 0.63 SR (NQO1/CPR) = 1.22
SR (L02 normal cells/A549 cancer cells) = 3.67
Poor selectivity to NQO1 and cancer cells
structure-selectivity
relationship
R2 O
R3 6 O
7 100 R1 = Me, R2 = H, R3 = Me, R4 = Me, R5 = H SR (NQO1/CPR) = 6.37
4 R 1 101 R1 = Me, R2 = Me, R 3 = H, R4 = H, R 5 = Me SR (NQO1/CPR) = 1.38
R 8 9
102 R1 = Me, R2 = H, R3 = Me, R4 = H, R 5 = H SR (NQO1/CPR) = 1.82
R5 O
2 103 R1 = H, R2 = H, R3 = Me, R4 = Me, R 5 = H SR (NQO1/CPR) = 2.51
modification
at the C2 site
of
improve the NQO1 activity
O O and selectivity
O O
ro
CH 3
CH3 H
H3 C N
H 3C R O O
H3 C
O
2
linker
-p
improve NQO1/CPR selectivity
H3C
104
N
re
improve water solubility
L-shaped NQO1 sustrates SR (NQO1/CPR) = 20.8
SR (L02 normal cells/A549 cancer cells) = 16.7
SR (A549 + DIC/A549 cancer cells) > 23.8
lP
Highly active and selective to NQO1 and cancer cells

with good water solubility (pH = 3) > 0.500 mg/mL
na
Fig. 13. Modifications of β-lap focusing on the NQO1/CPR selectivity and safety profiles.
ur
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Fig. 14. The docking poses of 100 (A) and 104 (B) in the active site of NQO1 (PDB ID: 2F1O). Blue
dot lines indicate hydrogen-bonding interactions, and purple dot lines indicate C-H...π interactions [59].
To further enhance the selectivity between NQO1/CPR, improve the catalytic efficiency, and
increase the water solubility of 100, we further introduced nitrogen-containing side chains on the C2
33
position of furan to fit with the L-shaped binding pocket of NQO1[59, 128]. Among them, 104 (Fig.13)
was identified as the most efficacy substrate for NQO1 with the highest reduction rate, kinetic
parameter, and selectivity ratio values of 1520 μmol NADPH/min/μmol NQO1, kcat/Km = 15 ± 3.8 ×
106 M-1 s-1 and 20.8, respectively [59]. In addition to CPR, 104 also showed good selectivity toward
other one-electron oxidoreductases, such as carbonyl reductase 1 (CBR1), thioredoxin reductase (Trx),
and carbonyl reductase (CBR). The docking studies between 104 and NQO1 suggested that the
L-shaped molecule could fully occupy the catalytic site of NQO1 and form π-stacking interactions with
of
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Phe232 and Tyr128 residues in the side binding pocket of NQO1 (Fig. 14B). The introduction of
-p
hydrophilic morphino moiety contributed greatly to its water solubility. In vitro studies of 104 indicated
re
that it was potent towards a panel of NQO1-overexpressing cancer cells, including the A549(NQO1+)
lP
Taxol-resistant cells, with IC50 between 2.1 and 4.9 μM, especially the (IC50 = 2.2 μM). Further in vivo
antitumor evaluation in A549 tumor xenografted nude mice model suggested that 104 (30 mg/kg: TGI
na
= 87.1%; 50 mg/kg: TGI = 90.3%) more active than β-lap (30 mg/kg: TGI = 78.5%; 50 mg/kg: TGI =
ur
84.9%). In addition, 104 (10 μM) was proved to be safe in view of no obvious hepatotoxicity on the
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zebrafish, while 1 showed significant hepatoxicity even at a much lower concentration of 1 μM. These
results emphasized that 104 possessed improved NQO1-targeting antitumor potency and better safety
profiles than β-lap, which was worthy of further development.
Recently, our group reported a novel chemotype with nonquinone scaffolds as NQO1 substrates
for NQO1-targeting cancer therapy, which could be regarded as an analog of β-lap with a different
redox cycling center (Fig. 15) [49]. The nonquinone hit compound (105) as a novel NQO1 substrate
containing A-, B-, C- and D-rings was discovered from a high-throughput screening on an in-house
compound library using the NADPH-based assay (Fig.15). Further modification and SAR studies
34
revealed the tricyclic compound (106) as the most efficient NQO1 substrate with a kcat/Km value of 6.2
± 1.1 × 106 M-1 s-1, and it was highly selective to A549/Taxol (NQO1+) cancer cells over normal liver
L02 cells (SR = 50.3), which were better than β-lap (kcat/Km = 4.4 ± 0.9 × 106 M-1 s-1; SR = 6.3).
Docking studies suggested that both the 105 and 106 oriented above the isoalloxazine ring of the bound
cofactor FAD as for hydride transfer by π-stacking interaction. In addition, the carbonyl group and N1
atom in the planar ring of both compounds could form interactions with Tyr126 and Tyr128 residues,
respectively (Fig. 16. A and B). Based on the binding modes, a possible redox cycling mechanism of
of
ro
the nonquinone substrates catalyzed by NQO1, as represented by 106, was reasonably proposed as
-p
shown in Fig. 16C. The C(9)=O moiety was observed as an electrophilic site that could receive hydride
re
(H−) from reduced FAD (FADH2) (Fig.16C); this was following that of quinone substrate β-lap (Fig. 16
lP
D). In vitro studies showed that 106 was highly potent toward A549/Taxol and A549 cell lines with
IC50 values of 0.6 and 1.0 μM, respectively. Further, the antitumor efficacy was evaluated against
na
A549/Taxol in vivo and the growth of A549/Taxol tumor was strongly inhibited by 106 (15 mg/kg: TGI
ur
= 33.4%, T/C = 62.1%; 30 mg/kg: TGI = 64.9%, T/C = 25.9%), which was more efficacious than Taxol
Jo
at 2 mg/kg (TGI = 15.2%, T/C = 82.4%). More importantly, no obvious changes in the weight of mice
treated by 106 were observed, emphasizing the preliminary safety profiles of 106 in vivo [49]. The
discovery of new chemotypes of nonquinone substrates for NQO1 may provide another interesting and
promising way for the modification of NQO1-targeting quinone compounds, such as β-lap.
35
of
ro
-p
re
lP
na
Fig. 15. The discovery of nonquinone analog of β-lap with NQO1-targeting antitumor activity. The unit
ur
for the NQO1 reduction rate is mmol NADPH/min/mmol NQO1.

Jo
36
of
ro
-p
re
lP
na
ur
Jo
Fig. 16. The conformations of compound 105 (A) and 106 (compound 20k; B) were docked to the
active site of NQO1 (PDB ID: 1H69). (C) The proposed redox cycling process for 106 (compound 20k)
by NQO1 reduction. (D) The probable reduction process for quinone substrate β-lap by NQO1 [49]
Reproduced from ref.42 with permission from the American Chemical Society, copyright 2018.
The in vitro antitumor activity of β-lap derivatives discussed above were all summarized in
Table1.
Table 1
Summary of the in vitro antitumor activities of β-lap derivatives.
37
Compound IC50/μM Cancer cell lines Refs
3 6.90-9.63 KB, HeLa, HepG2 [94]
4 0.92-3.22 KB, HeLa, HepG2 [94]
5 1.10-2.37 KB, HeLa, HepG2 [94]
6 7.61-24.13 KB, HeLa, HepG2 [94]
HSC3, SCC4, SCC9, SCC15, SCC25,
7 0.02-2.50 HepG2, HL-60, K562, AGP-01, [95]
ACP-02, ACP-03, HT-29, HCT-116
8 5.51-14.63 MCF-7, NCI-H292, HEP-2 [96]
9 2.26-6.81 MCF-7, NCI-H292, HEP-2 [96]
10 1.43-2.42 HL-60, MDAMB-435, HCT-8, SF-295 [97]
11 1.01-1.55 HL-60, MDAMB-435, HCT-8, SF-295 [97]
12 1.67-2.33 HL-60, MDAMB-435, HCT-8, SF-295 [97]
of
13 1.20-2.26 HL-60, MDAMB-435, HCT-8, SF-295 [97]
14 1.88-3.21 HL-60, MDAMB-435, HCT-8, SF-295 [97]
ro
15 0.41-1.59 PC-3, HCT-116, HL-60, MDAMB-435, SF-295 [98]
16 >8.48 PC-3, HCT-116, HL-60, MDAMB-435, SF-295 [98]
17 0.53-3.78
-p
HL-60, MOLT-4, HCT-116, HCT-8, PC-3, PC3M,
[99]
re
OVCAR-3, OVCAR-8, SF-295, MDAMB-435
18 1.80-5.05 [99]
lP

19 0.94-1.72 [99]
na
20 0.95-2.42 PC-3, HCT-116, SNB-19, MCF-7, B16F10 [100]

21 1.19-3.90 [99]
ur

22 1.11-2.23 [99]
Jo

23 1.75-3.89 [99]
24 1.22-2.81 [99]
25 0.37-1.48 HL-60, HCT-116, SF-295, NCI-H460, PC-3 [9]
HL-60, MDAMB-435, SF-295,
26 0.31-10 [101, 130]
HCT-8, PC-3, B-16, A549
27 1.38-3.44 HL-60, K562, MOLT-4, Jurkat [102]
28 1.11-7.7 HL-60, K562, MOLT-4, Jurkat, A549 [102, 130]
29 1.15-1.68 PC-3, HCT-116, HL-60, MDAMB-435, SF-295 [98]
30 23.92-110.34 Caco-2, Calu-3, MDAMB-231 [103]
31 0.46-13.25 MCF-7, NCI-H292, HEP-2 [96]
32 0.60-13.60 MCF-7, NCI-H292, HEP-2 [96]
33 1.19-4.79 HL-60, MDAMB-435, SF-295, HCT-8, PC-3, B-16 [101]
38
39 0.05-11.07 MOLT, CEM, KG1, K562 [104]
40 1.33-1.92 HL-60, K562, MOLT-4, Jurkat [102]
41 0.94-2.74 HL-60, K562, MOLT-4, Jurkat [102]
42 0.74-1.99 HL-60, K562, MOLT-4, Jurkat [102]
43 1.34-2.37 PC-3, HCT-116, HL-60, MDAMB-435, SF-295 [98]
44 2.59-3.35 PC3, HCT-116, HL-60, MDAMB-435, SF-295 [98]
45 0.23-0.64 HL-60, HCT-8, MDAMB-435, SF-295, B-16, PC-3 [105]
46 0.19-0.96 HL60, MDAMB435, HCT8, SF-295 [106]
47 0.20-0.93 HL-60, HCT-8, MDAMB-435, SF-295, B-16, PC-3 [105]
of
48 0.74-1.78 HL-60, MDAMB-435, HCT-8, SF-295 [106]
HL-60, HCT-8, MDA-MB-435, SF-295, B-16,
ro
49 0.17-0.56 [105]
PC-3
50 0.12-0.59 HL-60, HCT-8, MDA-MB-435, SF295, B-16, PC-3 [105]
51
52
0.15-0.39
0.34-1.05
-p
HL-60, MDAMB-435, HCT-8, SF-295
HL-60, HCT-116, SF-295, OVCAR-8
[106]
[107]
re
53 0.49-4.39 HL60, MDAMB435, HCT-8, SF-295 [106]
54 0.59-4.33 HL60, MDAMB-435, HCT-8, SF-295 [106]
lP
55 0.27-2.29 HL60, MDAMB-435, HCT-8, SF-295 [106]

56 0.33-0.88 HL-60, HCT-116, SF-295, OVCAR-8 [107]
na
57 0.23-0.70 HL-60, HCT-116, SF-295, OVCAR-8 [107]

58 0.16-0.89 HL-60, HCT-116, SF-295, OVCAR-8 [107]
ur
59 0.45-1.33 HL-60, HCT-116, OVCAR-8, SF-295 [108]

60 0.10-4.57 HL-60, HCT-116, OVCAR-8, SF-295 [108]
Jo
HL-60, HCT-116, PC-3, SF-295,

61 0.07-0.38 [109]
MDA-MB-435, OVCAR-8
HL-60, HCT-116, PC-3, SF-295,
62 0.07-0.24 [109]
MDAMB-435, OVCAR-8
HL-60, HCT-116, PC-3, SF-295,
63 1.06-2.52 [109]
MDAMB-435, OVCAR-8
64 2.12-4.15 PC-3, HCT-116, SNB-19, MCF-7, B16F10 [100]
65 0.9-4.2 SF-295, PC-3, HCT-116, K562 [110]
HL-60, OVCAR-8, MDAMB-435, SF-295, HCT-8,
PC-3, DU-145, MCF-7, MX1, HS578t, MOLT-4,
66 0.48-1.24 [111]
CEM, K562, SW-620, COLO-205, HS578t,
UACC-62, MDAMB-231, HCT-15, NCI-H358
PC-3, DU-145, HL-60, K562, MOLT-4,
Jurkat, HCT-116, HCT-8, SW620, MX-1,
67 0.34-2.17 [112]
HS578t, MDAMB-231, SF-295, SF-268,
MDAMB-435, UACC62, NCI-H460
68 0.28-0.81 PC-3, SF-295, MDAMB-435, SW620, HCT-116 [113]
39
69 0.92-0.94 HL-60, Jurkat [113]
70 0.22-1.18 HCT-116, HepG2 [114]
71 2.86 HepG2 [115]
CCRF-CEM, NCI-H23, COLO-205, SF-295,
72 0.2-1.86 LOXIMVI, [116]
OVCAR-5, SN12C, DU-145, MDAMB-435
CCRF-CEM, NCI-H23, COLO 205, SF-295,
73 0.19-2.56 LOXIMVI, [116]
OVCAR-5, SN12C, DU-145, MDAMB-435
74 2.625-8.143 HCT-116, HL-60, SF-295, NCI-1975 [117]
75 1.5 OCI-LY3 [118]
76 IC50=17.0 OCI-LY3 [118]
77 0.029-2.0 HBL-100, HeLa, SW1573, WiDr [119]
of
78 0.11-2.15 MDAMB-435, HL-60, HCT-8, SF-295 [120]
79 0.15-3.84 MDAMB-435, HL-60, HCT-8, SF-295 [120]
ro
80 0.17-3.40 MDAMB-435, HL-60, HCT-8, SF-295 [120]
81 0.21-3.21 MDAMB-435, HL-60, HCT-8, SF-295 [120]
82
83
0.56-3.06
0.42-3.01
-p
SNB-19, HCT-116, PC-3, MCF-7, HL-60
SNB-19, HCT-116, PC-3, MCF-7, HL-60
[121]
[121]
re
84 6.1 A549 [130]
85 7.0 A549 [130]
lP
86 9.2 A549 [130]

87 6.8 A549 [130]
na
88 16 A549 [130]
89 7.4 A549 [122]
ur
90 11 A549 [122]
91 4.9 A549 [122]
Jo
92 6.0 A549 [122]

93 4.7 A549 [122]
94 2.23-3.13 A549, MCF-7, HepG2 [133]
95 20 A549 [126]
96 19 A549 [126]
97 5.2 A549 [126]
98 4.6 A549 [126]
99 7.6 A549 [126]
100 2.4-9.3 A549, MCF-7, HepG2, HCT-116, H1299 [128]
101 2.9 A549 [128]
102 4.1 A549 [128]
103 8.5 A549 [128]
A549, MCF-7, HepG2, HCT-116,
104 2.1-4.9 [59]
H1299, HEK293, A549-Taxol
105 0.6-1.0 A549, A549-Taxol [49]
106 0.6-1.0 A549, A549-Taxol [49]
1 0.25-9.3 All above cell lines All above
40
Doxorubicina 0.02-1.28 All above cell lines All above
a
Reference antitumor drug.
3.2 Anti-T. cruzi derivatives
3.2.1.1 Dihydropyran derivatives
To discover novel chemotypes with potent trypanocidal activity based on the structure of β-lap, de
Castro et al. introduced aromatic substituents to the C2 site of C-ring, such as 107-113, and fused a 5-
or 6-membered cycloalkane ring onto C-ring, as represented by114-115 (Fig. 17) [11]. Among them,
of
ro
107 (IC50 = 56.0 μM), 108 (IC50 = 20.2 μM), 109 (IC50 = 51.4 μM), 114 (IC50 = 62.6 μM) and 115 (IC50
-p
= 35.4 μM) were 2-5 times more active than benznidazole (IC50 = 103.6 μM), the standard treatment for
re
Chagas disease, while 110-1113 showed comparable activity to this drug. The chloro substituted at the
lP
para position of the phenyl moiety, as in 108, was beneficial for improving the activity against T. cruzi.
na
For instance, 107 with an unsubstituted phenyl (IC50 = 56.0 μM) was less potent than 108. Introduction
of substituents, such as methyl (109, IC50 = 51.4 μM), methoxy (110, IC50 = 121.4 μM), fluorine (111,
ur
IC50 = 97.6 μM), and ethoxy (112, IC50 = 132.2 μM) to the para position of phenyl, were all found to be
Jo
less active when compared with 108.
Taking into account that 1,2,3-triazoles and molecular hybridization played key roles in drug
design [48, 134], de Castro group synthesized 10 (Fig. 17) introducing bromine and azide on the C3
site of C-ring of β-lap and further generating the triazole derivatives 11-14 by click chemistry [12].
Surprisingly, among them, the azide intermediate 10 (IC50 = 23.4 μM) was the most active against T.
cruzi, showing a 4-fold increase in activity in comparison with benznidazole (IC50 = 103.6 μM) and a
17-fold increase, comparing with its precursor, β-lap (IC50 = 391.5 μM). Although these triazole
derivatives, such as 11-14, were less active as compared to 10, they demonstrated higher activity than
41
β-lap, except for 12 (IC50 = 439.6 μM). Especially, 14 (IC50 = 106.1 μM) was three times more active
than β-lap. Given that triazoles may improve the physicochemical properties, such as water solubility, it
was suggested that the introduction of triazole moiety could be a promising strategy for further
optimization of the trypanocidal activity of β-lap.
In addition, da Silva Júnior et al. reported another type of triazole derivatives, such as
5-iodo-1,4-disubstituted triazole (15) and 1,5-disubstituted- 1,2,3-triazole (16) (Fig. 17) [98].
Interestingly, 15 (IC50 = 125.1 μM) exhibited increased activity as compared with its 1,4-regioisomer 11
of
ro
(IC50 = 313.0 μM), being approximately 3-fold more active than β-lap (IC50 = 391.5 μM) and
-p
comparable to benznidazole (IC50 = 103.6 μM). However, 16 with an additional iodo substituent to the
re
triazole moiety was considered to be inactive (IC50 > 1000 μM).
lP
In view of the potential ‘redox modulating’ properties of selenium-containing compounds and
various pharmacological activities of chalcogen-containing quinones [135-139], da Silva Júnior et al.

na
designed a series of sulfur- and selenium-containing derivatives of β-lap [100, 140]. However, most of
ur
the derivatives were moderately active or even inactive against T. cruzi except for 17 (IC50 = 54.9 μM),
Jo
20 (IC50 = 38.3 μM), 116 (IC50 = 102.9 μM) and 117 (IC50 = 109.2 μM) (Fig. 17). Compound 17 was
two times more active than benznidazole (IC50 = 103.6 μM), while 116-117 demonstrated similar
activity to that of benznidazole. Among them, 20 was the most active against T. cruzi with IC50 of 38.3
μM, which was 2.6 times more active than benznidazole (IC50 = 103.6 μM) [100].
42
of
ro
-p
re
lP
na
ur
Jo
Fig. 17. Dihydropyran derivatives of β-lap with anti-T. cruzi activity.
3.2.1.2 Dihydrofuran derivatives
To explore various modifications on the C-ring of β-lap, Ferreira et al. contracted the C-ring from
dihydropyran to dihydrofuran ring to obtain 26 (Fig. 18), and its IC50 value against epimastigote forms
of T. cruzi was reported as >50 μM [141]. de Castro et al. and de Lima et al. functionalized the furan
ring at C2 resulting in the linear alkyl and iodomethyl substituted dihydrofuran derivatives, as
represented by 27-28 and 118-119 (Fig. 18) [142, 143]. Compound 27 (IC50 = 398 μM) showed higher
activity against bloodstream trypomastigote forms of T. cruzi than 28 (IC50 = 641 μM), revealing that
43
the activity could be magnified by the introduction of an iodine atom [142]. Of these derivatives, 118
(IC50 = 20.7 μM) followed by 119 (IC50 = 46.8 μM) was the most active against the amastigote stage of
T. cruzi with the activity similar to that of benznidazole (IC50 = 19.93 μM) [143]. These results
illustrated that the lipophilicity increased by introducing the aliphatic substituents, probably leading to
better penetration at the plasmatic membrane of the parasite, was beneficial to the trypanocidal activity.
Importantly, 118 (LC50 = 2200 μM) and 119 (LC50 = 1700 μM) were less cytotoxic against LLCMK2
cells as compared with benznidazole (LC50 = 323.8 μM), suggesting that it would cause less potential
of
ro
side effects [143].
-p
Taking the unique biological activity of triazoles into consideration and aiming to find new
re
derivatives with potent activity against the parasite, da Silva Júnior et al. and de Castro et al.
lP
functionalized the dihydrofuran C-ring at the C2 or C3 sites of 26, and synthesized a series of triazole
derivatives based on the azide intermediates, such as 33, by Click chemistry, as represented by 120-124
na
(C2 modified) and 34-37, and 125 (C3 modified) (Fig. 18) [48, 140]. Among the triazole derivatives
ur
modified at the C2 site, 121-124 (IC50 of 52.9-89.5 μM) showed potent activity against the
Jo
trypomastigote form of T. cruzi. Especially, 122 (IC50 = 52.9 μM) and 123 (IC50 = 55.3 μM) were
two-fold more active than benznidazole (IC50 = 103.6 μM) [48]. Among the triazoles derivatives
modified at the C2 site, 33 (IC50 = 50.2 μM) and 36 (IC50 = 17.3 μM) were found to be significantly
active against trypomastigote forms of T. cruzi. Particularly, 36, with lipophilic phenyl attached to the
triazole moiety, was the most active probably due to its high lipophilicity that resulted in a better
penetration through the plasmatic membrane of the parasite [48]. While for 34, 35, and 37, the
hydroxyl group may be prone to convert into polar metabolites, which decreased the concentration and
hindered them to penetrate through the parasite plasmatic membrane, thus resulting in a much lower
44
trypanocidal activity. In addition, 125 was susceptible to be hydrolyzed due to the hemiacetal and the
intermediate could be eliminated in a similar way to that of 34, 35, and 37 [48]. Further, Silva group
synthesized new triazole derivatives, the 5-iodo-1,4-disubstituted and 1,5-disubstituted-triazoles, by
using the strategy of pharmacophore-based molecular hybridization [98]. Unfortunately, most of them
were found to be inactive (IC50 > 1000 μM) against trypomastigote forms of T. cruzi; only one
compound, 43 (Fig. 18), showed a moderate anti-T. cruzi activity with an IC50 value of 112.6 μM,
which was comparable to that of benznidazole (IC50 = 103.6 μM) [98]. Besides, da Silva Júnior et al.
of
ro
synthesized a series of triazole derivatives, as represented by 126 (Fig. 18), with the sugar moieties
-p
installed by click chemistry [140]. However, these derivatives (IC50 = 174.9-472.2 μM) were two to
re
four-fold less active than benznidazole (IC50 = 103.6 μM).
lP
As seen above, the C-ring modified derivative 36 with a phenyltriazole substituent at the C2 site
showed a promising trypanocidal activity (IC50 = 17.3 μM). Further modifications focusing on the
na
phenyl group of 36 were conducted by da Silva Júnior group [144]. Compounds 40-42, and 127-128
ur
(Fig. 18) with phenyl substituted by different electron-withdrawing or electron-donating groups were
Jo
synthesized for SAR investigation. Among them, 40 and 42 with fluro and nitro substituents, sustained
the potent activity against the trypomastigote form of T. cruzi, with IC50 values of 20.8 and 21.8 μM.
Comparing with 36, the introduction of methyl as in 128 (IC50 = 39 μM) and bromo as in 127 (IC50 =
101.5 μM) led to a slight to moderate decrease in potency, while the introduction of methoxy, as in 41
(IC50 = 359.2 μM) resulted in a greatly decreased activity. These results showed that the strong
electron-withdrawing groups such as p-F and p-NO2 on phenyl were beneficial for the activity,
probably due to the enhanced redox potential of the quinone structure, while strong electron-donating
groups, such as p-OMe, was detrimental to the activity [144].
45
By introducing another redox center, da Silva Júnior et al. designed and synthesized novel
derivatives, as represented by 129-130 (Fig. 18), through the hybridization of a para-naphthoquinone
with the dihydrofuran analog of β-lap [145]. Notably, 129 (IC50 = 6.8 μM) and 130 (IC50 = 8.2 μM)
presented 15-fold higher activity than that of benznidazole (IC50 = 103.6 μM). In addition, 130
presented weak cytotoxicity with the concentration resulting in damage of 50% of the mammalian cells
(LC50) of 281.6 μM toward the normal heart muscle cells, showing an SI of 34.3. The result highlighted
that 130 was worthy of further investigation.
of
ro
-p
re
lP
na
ur
Jo
Fig. 18. Dihdyrofuran derivatives of β-lap with anti-T. cruzi activity.
46
In view of the potent activity against methicillin-resistant bacterial strains of 131 (Fig. 19), de
Castro et al. continued the investigation to search novel prototypes with potent activity against T. cruzi
by introducing alkoxy groups at the C3 position of the dihydrofuran C-ring [12, 21]. Among the
C3-substituted alkyloxyquinones, 53 (IC50 = 47.2 μM) was the most active against the trypomastigote
forms of T. cruzi, being two-fold more active than benznidazole (IC50 = 103.6 μM). These results
indicated that alkoxy groups were beneficial for the trypanocidal activity [12].
de Castro et al. synthesized a series of arylamino derivatives modified at the C2 site, including the
of
ro
monosubstituted arylamino derivatives (45-47 and 132-134), and the disubstituted arylamino
-p
derivatives (48, 135-139) (Fig. 19) with by different electron-withdrawing or electron-donating groups
re
substituted on phenyl [12, 21]. Among the monosubstituted arylamines, only 45 (IC50 = 86.3 μM) and
lP
132 (IC50 = 88.2 μM) presented higher activity against T. cruzi than benznidazole (IC50 = 103.6 μM)
[21]. In the case of disubstituted arylamines, 135 (IC50 = 24.9 μM), 136 (IC50 = 43.8 μM), 137 (IC50 =
na
59.6 μM), and 48 (IC50 = 55.6 μM) were found to be two- to four-fold more active than benznidazole
ur
[12]. These results indicated that the electronic properties and positions of substituents affected greatly
Jo
their activity. For example, the introduction of an additional bromo or chloro was beneficial for the
activity. For instance, 47 (IC50 = 140.8 μM) and 133 (IC50 = 952.5 μM) were less active than
benznidazole, while 135 derived by introducing a second bromo substituent, turned out to be four times
more active than benznidazole, with an IC50 value of 24.9 μM. Similarly, the introduction of a second
chloro substituent based on 134 also enhanced the activity [12, 21]. In addition, the introduction of the
strong electron-withdrawing nitro group, such as o-NO2 (48, IC50 = 55.6 μM) or m-NO2 (45, IC50 = 86.3
μM), contributed significantly to the activity [12, 21].
47
Further, Ferreira et al. replaced the NH group with its bioisostere, a sulfur atom, to generate a
novel series of sulfur-containing derivatives (Fig. 19) [146]. All of them (IC50 of 9.2-182.7 μM) were
found to be more active than β-lap (IC50 = 391.5 μM). Especially, 65 (IC50 = 32.3 μM), 140 (IC50 = 9.2
μM), 141 (IC50 = 59.5 μM), and 142 (IC50 = 83.6 μM) were even more active than benznidazole (IC50 =
103.6 μM). Importantly, 140 with the highest activity showed weak cytotoxicity to the normal heart
muscle cells (LC50 = 38.89 μM). In general, the 3-phenylthio compounds showed higher activity as
compared to the 3-phenylamines compounds. For example, 140 was 40-fold more active than the
of
ro
corresponding arylamine 134 (IC50 = 384.4 μM). It suggested that the replacement of NH by S was
favorable for the activity against T. cruzi [21, 146]. -p

re
Considering that molecular hybridization was essential for medicinal chemistry and chalcones
lP
presented a wide spectrum of pharmacological activities [147, 148], da Silva Júnior et al. synthesized
five chalcone hybrids [140]. Unfortunately, most of them were inactive; the most active compound, 143
na
(Fig. 19) showed an IC50 value of 230.2 μM against the parasite.

ur
Jo
48
of
ro
-p
re
lP
na
ur
Fig. 19. Arylamino, alkoxy, and hybrid derivatives of β-lap modified at C-ring with anti-T. cruzi
Jo
activity.
3.2.1.3 Removal of C-ring
Aiming to investigate the importance of the C-ring of β-lap, Ferreira et al. synthesized a quinone
analog 144 (Fig. 20) with the removal of C-ring [141]. Although 144 (IC50 = 11 μM) showed potent
activity against epimastigote forms of T. cruzi (clone Dm28c), it was ten times less active than its
precursor, β-lap (IC50 = 0.9 μM). This emphasized that the C-ring attached to the ortho-naphthoquinone
scaffold (AB rings) played an important role in the activity of β-lap against the parasite.
49
Fig. 20. Anti-T. cruzi derivatives of β-lap missing A-ring.
3.2.2.1 Monocarbonyl modified derivatives
Taking into account that oxyran derivatives played an important role in trypanocidal agents, Ferreira et
of
ro
al. replaced the carbonyl of β-lap with an oxyran moiety [141]. However, the resulting compound 145
-p
(IC50 = 12 μM; Fig. 21) exhibited moderate activity against epimastigote forms of T. cruzi of the clone
re
Dm28c, being approximately 13 times less active than β-lap (IC50 = 0.9 μM). Nevertheless, it was less
lP
cytotoxicity against the normal Vero cells (LC50 > 50 μM) than β-lap (LC50 < 3.1 μM).
R
na
146 X= NH, R = Ph 149 X = O, R = Ph

N
O 147 X = NH, R = p-MeO-C6H4 150 X = O, R = O
X
O
O
ur
O O 151 X = O, R = p-MeO-C6H4
148 X = NH, R =
Jo
145 NH
Ph R4 R3
NH
N R5 R2
Ph
O
monocarbonyl N N R1
modification HO O
N NH
Ph
O
O O O
dicarbonyl 152 153
O modification
R1 R2
O
N 154 R1 = H, R2 = H, R3 = F, R4 = H, R5 = H
B N R3 155 R1 = Cl, R2 = H, R3 = H, R4 = H, R5 = H
156 R1 = H, R2 = H, R3 = Br, R4 = H, R5 = H
O
157 R1 = Me, R2 = H, R3 = H, R4 = H, R5 = H
O 158 R1 = H, R2 = Me, R3 = H, R4 = H, R5 = H
β-lapachone (1) 159 R1 = H, R2 = H, R3 = Me, R4 = H, R5 = H
160 R1 = n-hexyl, R2 = H, R3 = H
161 R1 = H, R2 = H, R3 = n-hexyl
50
Fig. 21. B-ring modified anti-T. cruzi derivatives of β-lap.
3.2.2.2 Dicarbonyl modified derivatives
Pinto et al. functionalized the carbonyl groups on B-ring leading to various heterocyclic
derivatives, which could be classified, structurally, into phenazine, cyclopentenone, indole,
naphthoimidazole, and naphthoxazole derivatives (Fig. 21) [149-152]. It was observed that: 1) most
naphthoimidazole derivatives (146-148) were more active than the corresponding naphthoxazole
derivatives (149-150) [151]. For example, 146 (IC50 = 37.0 μM) and 147 (IC50 = 259.3 μM) exhibited
of
ro
more potent activities than 149 (IC50 = 283.5 μM) and 151 (IC50 = 3502.5 μM) [151]. 2) Introducing an
-p
aromatic ring on the oxazole ring could improve the activity. For example, 149 (IC50 = 283.5 μM) with
re
a phenyl group and 150 (IC50 = 269.5 μM) with a methylenedioxyphenyl group on the oxazole ring
lP
were more active than the positive drug crystal violet (IC50 = 536.0 μM) [151]. 3) The introduction of
phenazine was an effective strategy to increase activity [153, 154]. For instance, the phenazine
na
derivative 152 (IC50 = 61.3 μM) showed nine-fold higher activity than that of crystal violet (IC50 =
ur
536.0 μM) [152]. 4) Other heterocyclic derivatives containing a cyclopentenone and indole moiety, as
Jo
represented by 153 (IC50 = 56.1 μM) and 148 (IC50 = 15.4 μM), respectively, were also found to be
much more potent than β-lap (IC50 = 391.5 μM) [151].
Based on the structure of 146 with phenyl substituent, de Castro et al. further investigated the
effects of various substituted phenyl groups, as represented by 154-159 (Fig. 21), on the activity
against T. cruzi [154]. Compared with the parent compound 146, 154-158 showed a slight decrease in
activity with IC50 values ranging from 37.5 to 98.0 µM [154]. Interestingly, 159, with a p-methylphenyl
group attached to the imidazole scaffold, exhibited significantly potent activity with an IC50 value of
15.5 μM, being approximately 4-fold more potent than its precursor, 146 [154].
51
As seen above, phenylimidazole derivatives, such as 146, 148, and 159, possessed potent anti-T.
cruzi activity. However, they were found to be severely toxic on host cells, thus limiting their further
development. Considering that the imidazole moiety acted as an important pharmacophore of this
chemotype against T. cruzi, [155-157], de Castro et al. further synthesized new imidazole derivatives
by introducing linear alkyl substituents at the nitrogen atom [158]. Of them, 160 (IC50 = 42.5 μM) and
161 (IC50 = 31.5 μM) (Fig. 21) exhibited the highest activity, which was two-fold and three-fold,
respectively, more active than benznidazole (IC50 = 103.6 μM), a standard drug against T. cruzi.
of
ro
Meanwhile, the LC50 values of 160-161 were 116.6 μM and 422.1 μM, with SI values of 2.7 and 13.4,
-p
respectively; it emphasized that 161 was much less toxic to mammalian cells.
The in vitro anti-T. cruzi activity of β-lap derivatives discussed above were all summarized in
re
Table2.
Table 2
lP
In vitro anti-T. cruzi activity of β-lap derivatives.

Compound IC50 (μM) Refs Compound IC50 (μM) Refs
na
10 23.4a [12] 118 86.7a [140]

a a
11 313.0 [12] 119 256.7 [48]
ur
a a
12 439.6 [12] 126 208.9 [140]
13 219.8a [12] 127 101.5a [144]
Jo
a a
14 106.1 [12] 128 39.6 [144]
a a
15 125.1 [98] 129 6.8 [145]
a a
16 >1000 [98] 130 8.2 [145]
a a
17 54.9 [140] 131 212.3 [21]
a a
20 38.3 [100] 132 88.2 [21]
b a
26 >50 [141] 133 952.5 [21]
a a
27 398 [142] 134 384.4 [21]
a a
28 641 [142] 135 24.9 [12]
a a
33 50.2 [48] 136 43.8 [12]
34 151.9a [48] 137 59.6a [12]
a a
35 348.1 [48] 138 284.3 [12]
a a
36 17.3 [48] 139 156.2 [12]
a a
37 57.8 [48] 65 32.3 [146]
a a
40 20.8 [144] 140 9.2 [146]
a a
41 359.2 [144] 141 59.5 [146]
a a
42 21.8 [144] 142 83.6 [146]
52
43 112.6a [98] 143 230.2a [140]
a b
45 86.3 [21] 144 11 [141]
46 857.3a [21] 145 12b [141]
a a
47 140.8 [21] 146 37.0 [151]
a a
48 55.6 [12] 147 259.3 [151]
a a
53 47.2 [12] 148 15.4 [151]
a a
107 56.0 [11] 149 283.5 [151]
a a
108 20.2 [11] 150 269.5 [151]
a a
109 51.4 [11] 151 3502.5 [151]
a a
110 121.4 [11] 152 61.3 [152]
a a
111 97.6 [11] 153 56.1 [151]
a a
112 132.2 [11] 154 98.0 [154]
113 122.9a [11] 155 39.4a [154]
of
a a
114 62.6 [11] 156 84.9 [154]
a a
115 35.4 [11] 157 90.8 [154]
ro
a a
116 102.9 [140] 158 37.5 [154]
a a
117 109.2 [140] 159 15.5 [154]
118
119
20.7
46.8
c
c
[143]
[143]
-p
160
161
42.5
31.5
a
a
[158]
[158]
re
b
0.9
120 213.5a [140] 1 [141, 151]
391.5a
lP
a d
103.6a
121 89.5 [140] Benznidazole [143, 158]
19.93c
na
122 52.9a [140] Crystal violetd 536.0a [151]

a
123 55.3 [140]
ur
a
Activity against trypomastigote forms of T. cruzi.
b
Activity against epimastigote forms of T. cruzi (clone Dm28c).
Jo
c
Activity against intracellular amastigote forms of T. cruzi.
d
Reference drug.
3.3 Anti-Mycobacterium tuberculosis derivatives
Ferreira group introduced aryl substituents on the C2 or/and C4 position of β-lap providing a series of
C-ring modified derivatives and evaluated their activity against M. tuberculosis H37Rv strain
(ATCC27294) [13]. All of them, as represented by 162-164 (Fig. 22) exhibited high inhibitory activity
with MICs between 1.25 to 6.25 μg/mL, being more active than the precursor β-lap (MIC = 12.5
μg/mL). SAR studies revealed that the introduction of the phenyl group on the C2 position of β-lap was
53
favorable for the inhibitory activity, while the presence of aryl substituents on the C4 position was
detrimental for it. For instance, 162 substituted with p-methylphenyl on the C2 site showed the highest
activity against ATCC27294 with MIC of 1.25 μg/mL, while 163 (MIC = 3.12 μg/mL) substituted with
phenyl on the C4 site reduced the inhibitory activity as compared to 164 (MIC = 2.5 μg/mL).
da Silva Júnior et al. synthesized triazole naphthoquinone derivatives by introducing phenyl
triazoles on the C4 position of the dihydropyran ring and evaluated their inhibitory activity against
ATCC27294 [14]. All the derivatives, as represented by 11-14, (Fig. 22), demonstrated effective
of
ro
inhibitory with MIC bellow 6.25 μg/mL, illustrating these derivatives were prone to be promising
-p
prototypes for further research. Subsequently, da Silva Júnior et al. introduced triazoles substituted with
re
various aryl, as seen in 36, 40-42, and 127-128 (Fig. 22), and linear alkyl groups, as seen in 165-168
lP
(Fig. 22) based on the dihydrofuran scaffold [14]. These triazoles were screened in the ATCC27294
strain; most of them showed moderate activity with MIC bellow 12.5 μg/mL. These results indicated
na
that dihydrofuran triazole derivatives showed lower activity than the corresponding dihydropyran
ur
triazole derivatives (11-14 with MIC bellow 6.25 μg/mL).

Jo
54
of
ro
-p
re
lP
na
ur
Jo
Fig. 22. Anti-Mycobacterium tuberculosis derivatives of β-lap.
The ortho-quinone group of β-lap was conveniently converted to heterocycles, such as imidazoles (148,
157, 159, 169-178) and oxazoles (179) (Fig. 22) by condensation reactions to generate a series of
B-ring modified derivatives with the inhibitory activity against ATCC27294, ATCC35338, and
ATCC35822 strains [22, 159]. Among the phenylimidazole derivatives, compounds with
electron-withdrawing groups substituted at the ortho-position, such as 169 (o-Br), 170 (o-NO2), and
55
171 (o-CF3), showed higher activities against all the three tested strains with MIC between 0.78-100
μg/mL when compared with the phenyl compound 172 (MIC > 100 μg/mL). Notably, 171 was
identified as the most active compound against ATCC27294, ATCC35338, and ATCC35822 strains
with MIC values of 1.56, 0.78, and 1.56 μg/mL, respectively [159]. Meanwhile, compounds with
electron-donating groups substituted on phenyl, such as 157 (o-Me) and 173 (o-OMe), were also active
against all the strains with MIC between 1.56 and 6.25 μg/mL. Among the meta-substituted derivatives,
174 (m-Br), 175 (m-F), and 176 (m-CF3) showed potent activities against all the strains with MIC
of
ro
values in the range of 1.56-6.25 μg/mL. As for para-substituted derivatives, 177 (p-NO2) showed the
-p
highest activity against the ATCC35822 strain with a MIC value of 1.56 μg/mL, while no activities
re
(MIC > 100 μg/mL) against the ATCC27294 and ATCC35338 strains were observed. In addition, 159
lP
(p-Me) was active against ATCC27294 and ATCC35338 strains with MIC of 3.12 μg/mL and 6.25
μg/mL, respectively [22]. Among the imidazole derivatives substituted with aromatic heterocycles, 178
na
with an isoquinoline moiety showed the highest activities against ATCC35338 and ATCC35822 strains
ur
with MIC values bellow 0.78 μg/mL, while 148 with an indole moiety was the most active against
Jo
ATCC27294 strain with a MIC value of 1.56 μg/mL [22, 159]. The oxazole derivatives showed
moderate to potent activity against all the strains. Among them, 179 was found to be the most active
compound against ATCC27294, ATCC35338, and ATCC35822 strains with MIC values of 6.25, 1.56,
and 3.12 μg/mL, respectively [159].
In addition, considering that the carbonyl group of B-ring was an important modification site, da
Silva Júnior et al. introduced hydrazine groups on the carbonyl group of β-lap to obtain a series of
hydrazone derivatives and evaluated their activity against three M. tuberculosis [160]. Most of these
derivatives remained active against M. tuberculosis. Among them, 180 was identified as the most
56
potent active against H37Rv with a MIC value of 0.78 µg/mL, which showed eight times higher
efficacy than its precursor β-lap (MIC = 6.25 µg/mL). It was worth noting that 180 (SI > 250) exhibited
lower cytotoxicity against H37Rv than β-lap (SI > 32) [160]. These results included that the
modification on B-ring could effectively improve the activity against M. tuberculosis and rarely
produce inactive compounds, emphasizing the rationality of this modification.
3.3.3 B- and C-ring modified derivatives
Almeida da Silva et al. and da Silva Júnior et al. synthesized a series of phenazine derivatives modified
of
ro
at B-ring with substituents such as alkyl, halo, or azido on the C-ring of β-lap and evaluated their
-p
inhibitory activities against ATCC27294 and ATCC35338 strains [14, 22]. These phenazine derivatives,
re
as represented by 181-183 (Fig. 22), showed moderate activities against both strains (MIC < 6.25
lP
μg/mL), being 181 the most active with MIC = 0.78 μg/mL [22]. Further research was carried out on
the phenazine derivatives by introduction azide, aryltriazole, or alkyltriazole on the C2 or C3 position

na
of the dihydropyran analog of β-lap by da Silva Júnior et al. [14]. These derivatives were screened in
ur
the ATCC27294 strain and unfortunately, all the derivatives were inactive with MIC > 100 μg/mL with
Jo
some exceptions. For example, 184 showed a moderate activity with a MIC value of 25 μg/mL.
Besides, da Silva Júnior and co-workers modified the C-ring based on the structure of 180 and
synthesized several derivatives [160]. Among them, 185 (MIC = 6.25 µg/mL, SI > 250) sustained a
similar activity as 180 against H37Rv with lower toxicity.
3.4 Antifungal and antibacterial derivatives
Studies on the antifungal ortho-quinones related to β-lap were performed mainly based on 26 (Fig. 23),
a dihydrofuran analog of β-lap. Ferreira group introduced aryl substituents on the C2 site or cycloalkyl
on the C-2 and C-3 position of the C-ring of 26, leading to some derivatives of 26; their inhibitory
57
activities were evaluated against two forms of fungi including S. schenckii and S. brasiliensis [16].
Most of the derivatives showed more than 70% inhibition against both the fungi at 32 μM. Among
them, 186 and 187 (Fig. 23) were found to be the most stable and efficacious compounds with MIC
values between 2 and 16 μM, being less active than itraconazole (positive control) with MIC values of
0.125 and 0.25 μM against S. schenckii and S. brasiliensis, respectively. Interestingly, 186-187 could
amplify the potency of itraconazole when they were combined with itraconazole against S. schenckii
and S. brasiliensis with MIC values of 0.06 and 0.03 μM, respectively.
of
ro
-p
re
lP
na
ur
Jo
Fig. 23. Antifungal derivatives of β-lap.
Castro et al. introduced triazole or arylamino substituents on the C3 position of 26 leading to two
groups of derivatives, including 1,2,3-triazoles and 3-arylamino derivatives, and their activities were
evaluated against E. faecalis multiresistant hospital strains [50]. Unfortunately, only the 1,2,3-triazole
derivatives demonstrated inhibitory activity against clinical E. faecalis strains, while the 3-arylamino
derivatives were inactive. Notably, 36 (MIC = 8 μg/mL) and 37 (MIC = 16 μg/mL) showed comparable
58
activity to the positive control, chloramphenicol (MIC = 12 μg/mL), suggesting that the introduction of
triazole moiety was favorable for the antibacterial profile.
dos Santos et al. synthesized a hydroxy derivative (131) of 26 and evaluated its inhibitory
activities against Staphylococcus species including S. haemolyticus MRSH 225, S. epidermidis MRSE
228, S. aureus ATCC33591 (MRSA), and S. aureus ATCC29213 (MSSA) [15]. Interestingly, 131
exhibited potent antibacteriostatic activities with MICs of 4-8 μg/mL and MBC more than 512 μg/mL;
131 also showed high cytotoxic effect against eukaryotic cells (BSC-40) with toxic concentration
of
ro
(minimal toxic concentration = 2 μg/mL) bellow MICs. Thus, it is necessary to reduce toxicity in
further modification. -p
re
In light of the microbicidal properties of sulfides and sulfones, Cavaleiro et al. introduced sulfur
lP
and sulfone substituents on the C3 position of dihydrofuran leading to two groups of derivatives,
leading to sulfur-containing compounds as represented by 65 and 188, and the sulfone-containing

na
compounds as represented by 189-190 (Fig. 23); their inhibitory activities were evaluated against both
ur
the Gram-negative (E. coli ATCC13706) and Gram-positive (S. aureus 2065 MA) bacteria [161].
Jo
Among the sulfur-containing compounds, most of them could inhibit the growth of Staphylococcus
aureus 2065 MA with growth inhibition halo diameters values of 8-11 mm; 65 and 188 were highly
potent with inhibition halos diameters of 11 mm and 10 mm, respectively. However, none of them were
active against Escherichia coli ATCC13706. In the case of the sulfone-containing compounds, similar
results were observed against Escherichia coli ATCC13706. Only 189 and 190 were found to be active
against Staphylococcus aureus 2065 MA with inhibition halos diameters of 7 and 8 mm, respectively.
3.5 Antimalarial derivatives
59
In view of the antimalarial activity of phenazines, Krettli group functionalized the B-ring leading to a
phenazine derivative 191 (Fig. 24) and evaluated its activity against four Plasmodium falciparum
strains - BHz26/86, HB3, D6, and W2 strains [18]. Unfortunately, 191 was observed as an inactive
compound with IC50 more than 20 μM. Further, Krettli group introduced alkyl substituent and sulfo
group onto the C3 position of 191 [18]. The derivatives were also evaluated against the four P.
falciparum strains. All of them exhibited moderate to potent activities with IC50 values ranging from
1.67 to 9.44 μM. Notably, 192 with a sulfonic acid group, was found to be the most active with IC50
of
ro
values in the range of 1.67-6.14 μM. Further in vivo studies revealed that 192 (200 mg/kg,
-p
subcutaneous) could effectively inhibit the growth of parasite with an inhibitory rate of 98% and reduce
re
significantly the mortality as compared with the non-treated control groups. These results indicated that
lP
192 exhibited potent activity against malaria both in vitro and in vivo, being a promising lead for
antimalarial therapy.
na
ur
Jo
Fig. 24. Antimalarial derivatives of β-lap.
4. Clinical studies of β-lap and its derivatives
As listed in Table 3, several clinical trials related to β-lap and its derivatives have been completed for
the treatment of cancer (both ARQ 510 and ARQ 761) and metabolic syndrome (MB12066) [44, 45,
60
162].
ARQ 501, an injection formation of β-lap with the addition of HP-β-CD, has entered seven
clinical trials including three Phase I trials [163-165], one Phase I/II trial [166], and three Phase II trials
[167-169] as monotherapy or in combination with gemcitabine for the cancer treatment, such advanced
solid tumors, head and neck neoplasms, squamous cell, pancreatic cancer, and adenocarcinoma. At the
dose bellow 390 mg/m2, ARQ 501 demonstrated apparent linear pharmacokinetics with a total body
clearance of 114 l/h/m2 and a steady-state volume of distribution of 490 l/m2, which were 2-fold and
of
ro
10-fold higher than hepatic blood flow and total body weight, respectively [162]. However, rapid
-p
elimination (t1/2 = 2.45 h) of ARQ 501 in the form of intravenous administration (1.5 mg/kg) from the
re
systemic circulation and low absolute bioavailability (15.5%) in the form of oral administration (40
lP
mg/kg) were suggested to be related to its poor water solubility, becoming the huge challenges in the
application of ARQ 501 [46]. A relatively high proportion (approx. 40%) of HP-β-CD was used in the
na
formation of ARQ501 to improve the water solubility of β-lap from 0.038 mg/mL to 16.0 mg/mL,
ur
enabling 400-fold enhancement of water solubility [125]. However, the occurrence of hemolytic
Jo
anemia was suggested to be in association with HP-β-CD in a long term treatment, thus hindering the
clinical application of ARQ 501 [47].
Subsequently, the company ArQule reported a water-soluble prodrug of β-lap (ARQ 761) due to
the poor water solubility of β-lap; the structure of ARQ 761 had not yet been disclosed [170]. ARQ 761
entered three Phase I trials (NCT01502800, NCT02514031, NCT03575078) as a single agent or in
combination with other antitumor drugs such as olaparib, nab-paclitaxel, or gemcitabine [171-173].
ARQ 761 presented a similar maximum tolerated dose (MTD) to ARQ 501 with MTD of 390 mg/m2 in
the form of 2 h intravenous administration every other week. Besides, ARQ 761 presented no
61
correlation with the HP-β-CD, a hydrophilic carrier leading to dose limited toxicity (DLT) hemolytic
anemia [44]. Only 20% of patients treated with ARQ 761 alone were observed in minor radiographic
responses, indicating the moderate potency of ARQ 761. In addition, a combination study with
nab-paclitaxel or gemcitabine (NCT02514031) was suspended due to quality testing necessary for the
study drug ARQ 761 [172]. A combination study with olaparib (NCT03575078) was withdrawn since
no human subjects were enrolled and no data regarding humans were collected or studied [173].
MB12066 (structure undisclosed), a derivative of β-lap, activated mitochondrial metabolism
of
ro
through the NQO1-mediated redox cycle leading to appetite inhibition and energy expenditure [19].
-p
MB12066 entered three Phase I trials (NCT01285388, NCT01444677, and NCT02338856) and one
re
Phase II trial (NCT02029586) [174-177]. At the single doses ranging from 10-400 mg, MB12066 was
lP
observed at its peak concentration with a Tmax of 0.5-10 h, and it disappeared from the systemic
circulation (fe < 1%) with T1/2 of 10.4-24.6 h [45]. Meanwhile, at the multiple doses ranging from 100
na
to 200 mg, MB12066 also presented well tolerance with T1/2 of 17.35 and 13.74 in 100 and 200 mg
ur
dose, respectively. The steady-state concentration of 100-mg dose group (Cmax = 3.87 μg/L, AUC =
Jo
29.92 μg⋅hour /L, accumulation index = 1.67) was similar to that of 200-mg dose group (Cmax = 3.82
μg/L, AUC = 31.13 μg⋅hour /L, accumulation index = 1.48). As for its adverse events (AEs),
gastrointestinal symptoms were the usual AEs of MB12066 both in the single (59%) and multiple (40%)
dose groups, and more than half occurred in the high-dose groups such as 300- and 400-mg single-dose
groups (58%) and 200-mg multiple-dose group (83%). Importantly, the gastrointestinal symptoms of
MB12066 could be recovered without treatment, indicating the safety of MB12066. These results
suggested that MB12066 possessed good safety profiles both in the single (10-400 mg) and multiple
(100-200 mg) dose groups. However, the Phase II trial (NCT02029586) was terminated without
62
recruitment on December 21, 2017 [177].
Table 3. Clinical trials of β-lap.
ClinicalTrials.go Phas
Drug Disease Status Start and end time
v Identifier e
Phase 2004.01.13-2009.04.2
NCT00075933 ARQ 501 Cancer Completed
I 8
Phase 2004.12.10-2009.04.2
NCT00099190 ARQ 501 Carcinoma Completed
I 8
Advanced Solid Phase 2007.09.03-2011.10.1
NCT00524524 ARQ 501 Completed
Tumors I 9
of
Phase
2008.02,22-2013.02.2
ro
NCT00622063 ARQ 501 Cancer I Completed
8
Phase
-p II
re
Pancreatic
ARQ 501, Cancer Phase 2005.02.02-2009.04.2
NCT00102700 Completed
lP
Gemcitabine Adenocarcinom II 9
a
Phase 2006.04.04-2009.04.2
na
NCT00310518 ARQ 501 Cancer Completed

II 9
Head and Neck
ur
Neoplasms, Phase 2006.08.01-2009.04.2

NCT00358930 ARQ 501 Completed
Carcinoma, II 8
Jo
Squamous Cell
Phase 2012.01.02-2020.03.1
NCT01502800 ARQ 761 Solid Tumors Completed
I 7
ARQ 761,
Gemcitabine,
Pancreatic Phase 2015.08.03-2019.10.1
NCT02514031 Suspended
Cancer I 8
Nab-Paclitaxe
ARQ761, Phase 2018.07.02-2019.09.2

NCT03575078 Lymphoma Withdrawn
Olaparib I 5
Metabolic
Phase 2011.01.28-2018.06.2
NCT01285388 MB12066, Syndrome, Completed
I 7
Obesity
63
Metabolic
MB12066, Phase 2011.10.03-2017.12.2
NCT01444677 Syndrome, Completed
Placebo I 1
Obesity
MB12066, Metabolic Phase 2015.01.14-2017.12.2
NCT02338856 Completed
Placebo Syndrome I 1
Nonalcoholic
MB12066, Phase Terminate 2014.01.08-2017.12.2
NCT02029586 Fatty Liver
Placebo II d 1
Disease
5. Conclusion
In this review, we attempt to give a comprehensive perspective of β-lap involving pharmacological
mechanisms, structural modifications, and related clinical trials. β-Lap has received extensive attention
of
ro
due to its numerous pharmacological activities, such as antitumor, anti-Trypanosoma cruzi,
-p
anti-Mycobacterium tuberculosis, antibacterial, antimalarial and anti-metabolic syndrome. This review
re
gave insight into the related pharmacological mechanisms of β-lap, especially its antitumor mechanism
lP
related to the NQO1-mediated redox cycling. Then, we provided a comprehensive understanding of its
na
structural modifications concerning different therapeutic potentials. Structure modifications were
systematically discussed with regard to the modifications on the A-, B-, C-rings of β-lap, and the
ur
derivatives of β-lap were classified according to the different modification sites and pharmaceutical
Jo
potentials. As for the NQO1-targeting antitumor derivatives, we provided an overview of the structural
modification strategies concerning the binding site of NQO1, the NQO1/CPR selectivity, and drug-like
properties for improving the NQO1-targeting antitumor potency and safety profiles. We also
summarized the clinical trials of β-lap and its derivatives including ARQ 501, ARQ 761, and MB12066.
This review might be inspiring for researchers to design and develop novel derivatives of β-lap,
especially targeting the NQO1 enzyme, with both the potent pharmacological activities and good safety
profiles.
Conflicts of interest
64
The authors declare that they have no conflicts of interest.
ORCID
Xiaojin Zhang: 0000-0002-1898-3071
Xiang Li: 0000-0002-4061-7115
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (Grant
of
81773571), Jiangsu Province Funds for Excellent Young Scientists (Grant BK20170088), and the
ro
Fundamental Research Funds for the Central Universities (Grant 2632019ZD14).
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re
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Highlights
• Pharmacological mechanisms of β-lapachone are included.

• Various modification strategies toward β-lapachone are provided.
• Numerous derivatives of β-lapachone are reviewed and classified.
• Derivatives undergoing clinical evaluation are summarized.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
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A comprehensive review on β-lapachone: Mechanisms, structural modifications, and

To appear in: European Journal of Medicinal Chemistry

Received Date: 18 August 2020

© 2020 Elsevier Masson SAS. All rights reserved.

Pharmaceutical University, Nanjing 211198, China

various pharmacological activities, such as antitumor, anti-Trypanosoma cruzi, anti-Mycobacterium

Keywords: β-lapachone; natural product; quinone derivatives; structural modification

ACC, Acetyl-CoA carboxylase; AEs, Adverse events; AMPK, Adenosine 5’-monophosphate-activated

DNA, Deoxyribonucleic acid; DUBs, Deubiquitinases; H2O2, Hydrogen peroxide; HP-β-CD,

inhibitory concentration; MTD, Maximum tolerated dose; NAD(P)H, Nicotinamide adenine

dinucleotide phosphate; NAD, Nicotinamide adenine dinucleotide; NQO1, NAD(P)H:quinone

oxidoreductase-1; PARP-1, poly(ADP-ribose)polymerase-1; PKA, Protein kinase A; PPARα,

specific protease 2; VEGFR2, Vascular endothelial growth factor receptor 2.

exhibited a variety of pharmacological effects, such as antitumor [8-10], anti-Trypanosoma cruzi

instance, based on β-lap with regard to its diverse bioactivity.

physicochemical properties and safety profiles [17, 22, 48-50].

are also summarized.

β-lap on metabolic syndrome [58].

Fig. 1. Pharmacological mechanisms of β-lap.

2.1 Antitumor mechanisms

2.1.1 β-Lap and NQO1

through the NQO1-meidated redox cycling.

repair, resulting in cell apoptosis and cell death[62].

Recently, β-lap was found to be an inhibitor of ubiquitin specific protease 2 (USP2) as a

2.1.2 β-Lap and topoisomerase

consequence, leading to DNA damage and cell apoptosis [78].

2.1.3 β-Lap and IDO1

biosynthesis of nicotinamide adenine dinucleotide (NAD), significantly aggravating the depletion of

2.1.4 β-Lap and telomerase

2.1.5 β-Lap and Hsp90 -p

raised by β-lap was also related to the Hsp90 pathway.

2.2 Mechanisms for the other biological activities

3. Structural modifications on β-lap

including antitumor, anti-Trypanosoma cruzi, anti-Mycobacterium tuberculosis, antibacterial, and

derivatives with their corresponding biological activities.

3.1 Antitumor derivatives

3.1.1 C-ring modified derivatives

3.1.1.1 Dihydropyran derivatives

Fig. 3. C-ring modified dihydropyran derivatives of β-lap with antitumor activities.

0.8-3) still needed to be improved [98].

da Silva Júnior et al. synthesized a series of selenium- or sulfur-containing derivatives modified at

contribute to the antitumor activity for the ortho-quinone derivatives of β-lap.

chemical type of β-lap derivatives with promising antitumor activity.

Fig. 4. C-ring modified dihydrofuran derivatives of β-lap with antitumor activities.

as a potential modification site. Camara et al. installed a hydroxymethyl or acetoxymethyl on the C3

cells, IC50 = 0.07 μM for MOLT cells) [104].

Using a molecular hybridization strategy, antitumor fragments such as para-naphthoquinone and

respectively [110]. These results emphasized the importance of selenium-containing pharmacophore in

antitumor mechanistic investigations [112, 113].

3.1.1.3 Tetrazolopyrimidine derivatives

naphthoquinone-fused tetrazolopyrimidines were designed and synthesized by Wu group [114, 115].

toward HepG2 cancer cells [115].

Fig. 7. Tetrazolopyrimidine derivatives of β-lap with antitumor activity.

3.1.3 A-ring modified derivatives

3.1.4 A- and C-ring modified derivatives

hydroxypropyl-β-cyclodextrin (HP-β-CD) was required for the formulation for intravenous

substrates, such as β-lap.

hydrogen-bonding interactions [126].

improved NQO1/CPR selectivity (SR(NQO1/CPR) = 1.38-6.37) compared with that of 89

residues, respectively (Fig. 14A) [59].

Highly active and selective to NQO1 and cancer cells

profiles than β-lap, which was worthy of further development.