**Naturally Occurring Calanolides: Occurrence, Biosynthesis, and Pharmacological Properties Including Therapeutic Potential**

**Lutfun Nahar 1,\*, Anupam Das Talukdar 2 , Deepa Nath 3 , Sushmita Nath 4 , Aman Mehan 5 , Fyaz M. D. Ismail 4 and Satyajit D. Sarker 4,\***

Academic Editors: Maria João Matos and Pascal Richomme Received: 30 September 2020; Accepted: 26 October 2020; Published: 28 October 2020

**Abstract:** Calanolides are tetracyclic 4-substituted dipyranocoumarins. Calanolide A, isolated from the leaves and twigs of *Calophyllum lanigerum* var. *austrocoriaceum* (Whitmore) P. F. Stevens, is the first member of this group of compounds with anti-HIV-1 activity mediated by reverse transcriptase inhibition. Calanolides are classified pharmacologically as non-nucleoside reverse transcriptase inhibitors (NNRTI). There are at least 15 naturally occurring calanolides distributed mainly within the genus *Calophyllum,* but some of them are also present in the genus *Clausena*. Besides significant anti-HIV properties, which have been exploited towards potential development of new NNRTIs for anti-HIV therapy, calanolides have also been found to possess anticancer, antimicrobial and antiparasitic potential. This review article provides a comprehensive update on all aspects of naturally occurring calanolides, including their chemistry, natural occurrence, biosynthesis, pharmacological and toxicological aspects including mechanism of action and structure activity relationships, pharmacokinetics, therapeutic potentials and available patents.

**Keywords:** calanolides; pseudocalanolides; calanolide A; *Calophyllum*; Calophyllaceae; anti-HIV; reverse transcriptase; non-nucleoside reverse transcriptase inhibitors (NNRTIs)

#### **1. Introduction**

Calanolides are tetracyclic 4-substituted dipyranocoumarins, and their C-ring contains a *gem*-dimethyl group (Figure 1), e.g., (+)-calanolide A (**1**), (−)-calanolide B (costatolide) (**14**) (Figure 2). The discovery of calanolides from the leaves and twigs of the tree *Calophyllum lanigerum var. austrocoriaceum* (Whitmore) P. F. Stevens, collected from Sarawak, Malaysia in 1987 happened during one of the largest anti-HIV screening programs conducted by the National Cancer Institute (NCI) during 1987–1996. In that program, over 30,000 plant extracts were screened utilizing an in vitro cell-based anti-HIV screen that could determine the degree of HIV-1 replication in treated infected lymphoblastic cells versus that in treated uninfected control cells [1,2]. Calanolide A (**1**) (Figure 1), which can be described as a 11,12-dihydro-2*H*,6*H*,10*H*-dipyrano[2,3-f:2′ ,3′ -h]chromen-2-one substituted by a hydroxyl (–OH) group at C-12, methyl groups at positions 6, 6, 10 and 11 and a propyl group at C-4

(the 10*R*,11*S*,12*S* stereoisomer), was isolated as the first member of anti-HIV compounds, calanolides, as a potential novel therapeutic option for the treatment of HIV infections. However, a subsequent attempt to recollect this plant sample failed and the collection of other specimens of the same species (not necessarily the same variety), afforded only a negligible amount of calanolide A (**1**). In fact, calanolides are among the first plant-based compounds to demonstrate potential anti-HIV-1 activity. Later, an extract of the latex of *C. teysmanii* showed significant anti-HIV activity in the screening, but the major active compound was (−)-calanolide B (**14**, also known as costatolide), regrettably not calanolide A (**1**) (Figure 2). The anti-HIV activity of (−)-calanolide B (**14**) was less potent than that of calanolide A (**1**), possibly because of difference in stereochemistry at the chiral centers. To date calanolides A-F and some of their methyl, acetyl and dihydro derivatives have been reported mainly from various *Calophyllum* species (Figure 2). Among these, the structures of calanolides C (**6**) and D (**7**), as reported initially by Kashman et al. [1] from *C. lanigerum*, were revised and renamed as pseudocalanolides C (**8**) and D (**9**) [3] (Figure 2). However, the true calanolides C (**6**) and D (**7**) were later reported from *C. brasiliense* Cambess. [4–6].

**Figure 1.** Rings A, B, C and D, and carbon numbering in (+)-calanolide A (**1**).

The first isolation of calanolides from *C. lanigerum var. austrocoriaceum,* involved multiple steps, starting with the extraction of dried fruits and twigs of this plant with a 1:1 mixture of dichloromethane and methanol, followed by a sequential solvent partitioning process involving various solvents. The *n*-hexane and CCl4 fractions emerged as the active fractions [1]. Repeated vacuum liquid chromatography (VLC) on silica gel, eluting with a mixture of *n*-hexane and ethyl acetate afforded crude calanolides, which were further purified by HPLC, employing normal phase for calanolide A (**1**), calanolide B (**4**) and pseudocalanolide D (**9**) [reported incorrectly as calanolide D (**7**)], while reversed-phase for 12-acetoxycalanolide A (**2**), 12-methoxycalanolide A (**3**), 12-methoxycalanolide B (**5**), pseudocalanolide C (**8**) [reported as calanolide C (**6**)] and calanolide E (**10**). The structures of these compounds were determined by a combination of UV, IR, NMR and MS spectroscopic methods, and all spectroscopic data were published [1]. The absolute stereochemistry of calanolides A (**1**) and B (**4**) was confirmed by a modified Mosher′ s method.

There is a review [7] and a book chapter on calanolides [8], published about six years ago, that mainly cover anti-HIV activity, and the literature published until early 2014. This present review is not on the genus *Calophyllum*, the family Calophyllaceae or pyranocoumarins a such, but it exclusively focuses on various aspects of naturally occurring calanolides. This review is significantly different from any other previous articles on calanolides in its approach and coverage, and is a comprehensive update on naturally occurring calanolides, encompassing their chemistry, natural occurrence, biosynthesis, pharmacological and toxicological aspects including mechanism of action and structure activity relationships, pharmacokinetics, therapeutic potentials and available patents.

**Figure 2.** *Cont.*

**Figure 2.** Naturally occurring calanolides

#### **2. Occurrence**

Calanolides, calanolide A (**1**) being the first member of these 4-substituted pyranocoumarins isolated from *C. lanigerum var. austrocoriaceum*, are almost exclusively distributed within the genus *Calophyllum* L., which comprises a large group of ca. 200 species of tropical trees distributed in the Indo-Pacific region, but was also reported from one species (*Clausena excavate* Brum. f.) of the closely related genus *Clausena* [9–12] (Table 1). Calanolide A (**1**) and other calanolides were subsequently isolated from other *Calophyllum* species, e.g., *C. brasiliense* Cambess. [4,13,14], *C. inophyllum* L. [6], *C. teysmanii* Miq. [2] and *C. wallichianum* Planch. & Triana [15]. In a chemotaxonomic study on the *Calophyllum* species, the presence of calanolides was detected in the extracts of *C. inophyllum*, *C. lanigerum* var. *austrocoriaceum*, *C. mole* King, *C. nodosum*, aff. *Pervillei* Vesque., *C. soulattri* Burm. f., *C. tacamahaca* Willd. and *C. teysmanii* [9] (Table 1).

**Table 1.**Naturally occurring calanolides, their sources and properties.

**Table 1.** *Cont.*

Bernabe-Antonio et al. [5] reported the production of calanolides in a callus culture of *C. brasiliense*, where different concentrations and combinations of plant growth regulators were tested in leaf and seed explants to establish callus cultures capable of producing calanolides. Higher calanolides B (**4**) and C (**6**) production was observed in calluses from seed explants than those developed from leaves. In continuation of the search for new natural anti-HIV compounds, and at the same time to find new botanical sources of calanolides, McKee et al. [20] purified calanolide E2 (**12**), and calanolide F (**13**) from the extracts of *C. lanigerum var. austrocoriaceum* and *C. teysmanii var. inophylloide* (King.) P. F. Stevens. Later, costatolide (**14**), also known as (−)-calanolide B, was reported as an anti-HIV compound present in *C. cerasiferum* Vesque and *C. inophyllum* L. [24]. Calanolides A (**1**), and C (**6**), and costatolide (**14**) were isolated from the leaves of *C. brasiliense*, and their anti-HIV potential was evaluated [4].

#### **3. Biosynthesis**

Calanolides are biosynthesized from the parent simple coumarin 7-hydroxycoumarin, also known as umbelliferone (Schemes 1–3). The biosynthesis of umbelliferone in plants starts from the amino acid L-phenylalanine, and proceeds through the formation of *trans*-cinnamic acid, *p*-coumaric acid, 2-hydroxy-*p*-coumaric acid, 2-glucosyloxy-*p*-coumaric acid, and 2-glucosyloxy-*p*-*cis*-coumaric acid with the help of various enzymes like cinnamate 4-hydroxylase, 4-coumarate-CoA ligase, 4-coumaroyl 2 ′ -hydroxylase and so on [27]. The biosynthesis of dipetalolactone, a pyranocoumarin, and subsequent conversion to the 3-propyl-intermediate for calanolides may proceed through two routes, one through conversion of umbelliferone to osthenol (Scheme 1), and the other via formation of 5,7-dihdroxycoumarin (Scheme 2). Reactions are generally mediated by p450 monooxygenase and other non-p450 enzymes [28]. 3-Propyl-intermediate is converted to the precursor compound for calanolides A–C (**1, 4** and **6**), utilizing the Wagner-Meerwein rearrangement reaction, and the precursor compound is believed to be converted to calanolides with the help of p450 monooxygenase enzyme (Scheme 3). Published studies on the biosynthesis of calanolides are rather limited and only two publications are available on this topic to date [28,29]. Therefore, detailed knowledge of specific enzymes involved in the biosynthesis of calanolides is still in its infancy.

**Scheme 1.** Plausible biosynthetic route to 2′ -hydroxydihydrodipetalolactone from umbelliferone *via* formation of 5-hydroxy-6-prenylseselin.

**Scheme 2.** Plausible biosynthetic route to 2′ -hydroxydihydrodipetalolactone from umbelliferone *via* formation of 8-prenylalloxanthoxyletol.

In a recent study, the influence of soil nutrients, e.g., Ca2+ and K+, on the biosynthesis of pharmacologically active calanolides in the seedlings of *C. brasiliense* was studied [29]. It was observed that the use of K+ deficient modified Hoagland solution (MHS) could induce a 15, 4.2 and 4.3-fold decrease of calanolides B (**4**), C (**6**), and apetalic acid concentrations in the leaves of the seedlings, respectively. On the other hand, Ca2+ deficient MHS could lead to a decrease of 4.3 and 2.4-fold for calanolides B (**4**) and C (**6**), respectively. This study demonstrated that, like many other plant secondary metabolites, the biosynthesis of calanolides, albeit genetically controlled, may also be affected by environmental conditions, e.g., soil nutrients (minerals).

As genes dictate biosynthesis of secondary metabolites, a study was conducted to identify candidate genes that regulate to the biosynthesis of calanolides in *C. brasiliense* [28]. The unigene dataset constructed in this study could offer an insight for further molecular studies of *C. brasiliense*, particularly for characterizing candidate genes responsible for the biosynthesis of angular and linear pyranocoumarins. The candidate genes, e.g., UN36044, UN28345 and UN34582, identified in the transcriptome of the leaves, stem and roots of *C. brasiliense* might be involved in the biosynthesis of calanolides, which are essentially modified angular pyranocoumarins. Candidate unigenes in the transcriptome dataset were screened using mainly homology-based BLAST and phylogenetic analyses. It is worthy of mention that the BLAST programs are widely used for searching protein and DNA databases for optimizing sequence similarities [30]. For protein comparisons, several definitional, algorithmic and statistical refinements allow substantial decrease in the execution time of the BLAST programs and enhancement of their sensitivity to weak similarities.

**Scheme 3.** Plausible biosynthetic route to calanolides A (**1**), B (**4**) and C (**6**) from the intermediate, 2 ′ -hydroxydihydrodipetalolactone.

#### **4. Pharmacological Properties**

Although well-known for non-nucleoside reverse transcriptase inhibitory activity offering anti-HIV potential, calanolides have also been shown to possess various other pharmacological properties (Figure 3). The following sub-sections deal with anticancer, anti-HIV, antimycobacterial and antiparasitic activity of naturally occurring calanolides. As much of the published pharmacological studies, both in vitro and in vivo including human trials, on naturally occurring calanolides are about their anti-HIV property, over the years, significant amounts of information have become available on their mechanism of action, structure-activity-relationships, synergistic and/or additive property and their potential in anti-HIV combination therapy, which have been discussed adequately under individual headings within the anti-HIV sub-section. All other pharmacological properties of these compounds as outlined in different publications still require further investigations to establish their realistic therapeutic potential. Also, in silico pharmacological activity and toxicity studies on these pyranocoumarins have just begun to emerge in recent years.

**Figure 3.** A pictorial summary of pharmacological properties of naturally occurring calanolides.

#### *4.1. Anticancer Activity*

In the later part of 1980s, as a part of the initiative of the United States National Cancer Institute (NCI), plant samples from the Malaysian flora were collected for routine screening for potential cytotoxicity against a collection of cancer cell lines as well as for possible anti-HIV activity. One of the samples, the leaves and twigs of the tree *C. lanigerum* var. *austrocoriaceum*, despite not being active against any of the cancer cell lines tested, showed inhibitory activity of viral replication when tested against HIV-1 virus [31,32]. However, later, calanolide A (**1**) and calanolide C (**6**) were shown to possess antiproliferative or antitumor-promoting property through inhibition of TPA-induced EBV-EA activation in Raji cell lines [13]. The phorbol ester, 12-*O*-tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation and apoptosis in myeloid leukemia cells. Calanolide A (**1**) was found to be more active (IC50 = 290 mol ratio/32 pmol TPA) than its 10,11-*cis*-isomer, calanolide C (**6**) (IC50 = 351 mol ratio/32 pmol TPA). It was inferred that 4-substituted pyranocoumarins like calanolides might possess potential as cancer chemopreventive agents or antitumor-promoters. A recent study with the crude ethanolic extract of the leaves of *C. inophyllum* revealed its potential as a cytotoxic agent (IC50 120 µg/mL) against the breast cancer cell line MCF-7 [33]; it was also found to possess antiproliferative and apoptotic properties. However, no definitive proof was provided to establish which of the secondary metabolites biosynthesized by this plant, calanolides being one major class, were responsible for the putative anticancer activity. Although not calanolides, a few other 4-substituted coumarins, isolated from *C. brasiliense*, were tested against human leukemia HL-60 cells with some promising results [34], which might highlight the need for more comprehensive studies with all major 4-susbtitued coumarins, including calanolides, to find antileukemia lead compounds for new anticancer drug development. Calanolide A (**1**), isolated from a chloroform extract of *Clausena excavata*, was found to induce toxicity to the cells used in a syncytium assay for anti-HIV activity [10].

The efficacy of calanolide A (**1**) in AIDS-associated cancer was evaluated in silico utilizing an integrated approach combining network-based systems biology, molecular docking and molecular dynamics [35]. Molecular targets were screened and only the targets, e.g., HRAS, that are common to HIV and sarcoma, HIV and lymphoma, and HIV and cervical cancer, were utilized in this study. Calanolide A (**1**) was found to form a stable complex with the screened target HRAS, which is a small G protein in the RAS subfamily of the RAS superfamily of small GTPases, and is considered as a proto-oncogene; when mutated, this proto-oncogene has the potential to cause normal cells to become cancerous.

#### *4.2. Anti-HIV Activity*

Calanolide A (**1**), an anti-HIV non-nucleoside reverse transcriptase inhibitor (NNRTI), paved the way for the discovery and synthesis of a series of 4-substituted angular pyranocoumarins with potential anti-HIV property [1,36]. NNRTIs are a class of anti-HIV drugs that prevent healthy T-cells in the body from becoming infected with HIV. Kashman et al. [1] first reported this new class of anti-HIV agents from the tropical rainforest tree, *C. lanigerum*. Calanolide A (**1**), 12-acetoxycalanolide A (**2**), 12-methoxycalanolide A (**3**), calanolide B (**4**), 12-methoxycalanolide B (**5**), pseudocalanolide C (**8**), pseudocalanolide D (**9**) and calanolide E (**10**) (Figure 2) were isolated through an anti-HIV bioassay-guided isolation. Calanolides A (**1**) and B (**4**) were found to be protective against HIV-1 replication and cytopathicity with EC50 values of 0.1 µM and 0.4 µM, respectively. However, both compounds were inactive against HIV-2, which is known as less pathogenic than HIV-1 and mainly found in West African countries. The other compounds showed a low level of anti-HIV-1 activity. This study involving purified bacterial recombinant reverse transcriptases established that the calanolides are indeed HIV-1 specific reverse transcriptase inhibitors. A comparative report on the anti-HIV potentials of calanolide A (**1**), costatolide (**14**) and dihydrocostatolide (**16**) against a series of HIV isolates of different cellular phenotypes was published by Buckheit et al. [26], which clearly demonstrated that calanolide A (**1**) was the best anti-HIV candidate among the three calanolides tested.

Two analogs of calanolide A (**1**), i.e., costatolide (**14**) and dihydrocostatolide (**16**), were shown to possess anti-HIV property similar to that of calanolide A (**1**) [26] and could be ascribed to the class of NNRTIs. In fresh human cells, costatolide (**14**) and dihydrocostatolide (**16**) could significantly inhibit the low-passage clinical virus strains, including those representative of the various HIV-1 clade strains, syncytium-inducing and non-syncytium-inducing isolates, and T-tropic and monocyte-tropic isolates [26,37]. In continuation of the search for new natural anti-HIV compounds, McKee et al. [20] purified calanolide E2 (**12**), and calanolide F (**13**) from extracts of *C. lanigerum* var. *austrocoriaceum* and *C. teysmanii* var. *inophylloide* (King.) P. F. Stevens, and calanolide E2 (**12**) emerged as one of the most active anti-HIV compounds. Later, costatolide (**14**) was reported as an anti-HIV compounds present in *C. cerasiferum* Vesque and *C. inophyllum* L. [24], while calanolides A (**1**), and C (**6**), and costatolide (**14**), isolated from the leaves of *C. brasiliense*, were shown to possess anti-HIV potential [4]. Comparative anti-HIV activities of some naturally occurring calanolides, e.g., calanolide A (**1**), costatolide (**14**) and dihydrocostatolide (**16**), against various strains of HIV are available in the article by Buckheit, et al. [26].

#### 4.2.1. Activity Against Drug Resistant Strains of HIV-1

Interestingly, calanolide A (**1**) was not only found to be active against standard strains of HIV-1, but it was also active against the resistant strains, eAZT-resistant G-9106 strain of HIV-1 and pyridinone-resistant A17 strain [1,38]. The activity against the pyridinone-resistant A17 strain was of interest as this strain is highly resistant to most of the HIV-1 specific NNRTIs, for example, TIBO, BI-RG-587 and L693,593. Later, it was established that pyranocoumarin **1** could interact with HIV-1 reverse transcriptase within the previously defined common binding site for nonnucleoside inhibitors [38]. An assessment of the inhibition patterns of the chimeric reverse transcriptases containing complementary segments of HIV-1 and HIV-2 reverse transcriptases established that there was a segment between residues 94 and 157 in HIV-1 reverse transcriptase that was crucial for inhibition by calanolide A (**1**) [39]. However, it was assumed that there might be a second

segment, essential for specifying susceptibility to the drug, between amino acids 225 and 427 in HIV-1 reverse transcriptase. A couple of years later, it was noted that calanolide A (**1**) was active against virus isolates resistant to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine and its derivative, [1-benzyloxymethyl-5-ethyl-6-(alpha-pyridylthio)uracil] [40]. Furthermore, this pyranocoumarin (**1**) showed activity against HIV with the two most common NNRTI-related mutations, K103N and Y181C, and was found to select for a mutation that does not cause cross-resistance with any other NNRTIs under investigation. It was postulated that substitution at codon Y188H of reverse transcriptase could be associated with 30-fold resistance to calanolide A (**1**) in vitro [41]. The compound is essentially inactive against all strains of the less common HIV type 2. It is necessary to carry out appropriate in vivo experimentations, either in animal models or in human clinical trials, to understand the true potential of any putative drug candidate. In vivo anti-HIV activity of (+)-calanolide A (**1**) was assessed in a hollow fibre mouse model [42], and it was observed that this compound could suppress virus replication in two unique, but separate physiologic compartments following oral or parenteral administration.

Calanolides were found to possess an enhanced antiviral activity against one of the most prevalent NNRTI-resistant viruses that is engendered by the Y181C amino acid change in reverse transcriptase as well as with reverse transcriptases that possess the Y181C change together with AZT-resistant mutations [26,37]. Calanolides could also be active against viruses containing Y181C and K103N dual mutations, which are generally highly resistant to other known non-nucleus reverse transcriptase inhibitors. Anti-HIV activity of naturally occurring calanolides against drug-resistant strains of HIV have made these compounds promising structural templates for new anti-HIV drug development.

#### 4.2.2. Calanolides in Anti-HIV-1 Combination Therapy

For the treatment of HIV infections, use of combination therapy comprising several anti-HIV drugs has become a common practice in recent years. The synergistic effects of calanolide A (**1**), costatolide (**14**) and dihydrocostatolide (**16**) [26] in combination with established anti-HIV drugs, e.g., azidothymidine (AZT), indinavir, nelfinavir and saquinavir, are available in the literature [26]. Synergistic effects were observed in both cultured cells and animal models when calanolides were used in combination with other anti-HIV agents [43]. Both calanolide A (**1**) and costatolide (**14**) were found to be effective in combination therapy for HIV infections [44]; in combination with NNRTIs, costatolide (**14**) could only synergistically inhibit HIV type 1 with UC38, whilst calanolide A (**1**) in combination with one of the NNRTIs helped this drug to retain activity against virus isolates with the single Y181C mutation [26,41,44,45].

A combination of (+)-calanolide A (**1**) and nevirapine (marketed under the trade name viramune among others for the treatment and prevention HIV-1 infection) was found to possess an additive to weakly synergistic effect in blocking replication of HIV-1 in an in vitro tissue culture assay [41], indicating the possibility of using (+)-calanolide A (**1**) in anti-HIV-1 combination therapy. In an in vivo study using a hollow fibre mouse model [42], the synergistic potential of (+)-calanolide A (**1**) in combination therapy with AZT, a well-known anti-retroviral medication, was further established. A more comprehensive study on the anti-HIV activity of (+)-calanolide A (**1**) and its analogs, e.g., costatolide (**14**), dihydrocostatolide (**16**) and (+)-12-oxo-calanoldie A, in combination with other inhibitors of HIV-1 replication was published about a decade ago [37,46]. Calanolides were found to display synergistic antiviral interactions with other nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors. In addition, additive interactions were also observed with calanolides when used with other anti-HIV drugs. It was concluded that the utility of convergent and divergent combination therapies using reverse transcriptase inhibitors and protease inhibitors in combination with (+)-calanolide A (**1**) or one of its analogues could be clinically relevant. Budihas et al. [47] demonstrated significant synergy between β-thujaplicinol and calanolide A (**1**).

#### 4.2.3. Structure-Activity-Relationships (SAR)

Among the naturally occurring calanolides, calanolide A (**1**) is one of the most potent anti-HIV compounds and has been the focus of various studies including the study of its possible mechanism of action, structural modifications, pharmacokinetics and toxicity [9,48–51]. The structures of naturally occurring calanolides mainly differ in their stereochemistry at various chiral centers (C-10, C-11 and C-12) on the ring D (Figures 1 and 2). McKee et al. [20] reported that calanolide-type compounds with a 12β hydroxyl group (as in compound **1**) generally possess anti-HIV activity. While calanolide A (**1**) and costatolide (**14**) were found to be active, (+)-calanolide C (**6**) was inactive in the in vitro anti-HIV assay [4,24]. The inactivity of (+)-calanolide C (**6**) despite possessing the pharmacophoric ring D, as well as a propyl group on C-4, could be due to the β-*cis* orientation of methyl groups on C-10 and C-11.

Like any other optically active drug molecules, optical activity plays an important role in the anti-HIV activity of calanolides. It has long been established that (+)-calanolide A (**1**) and (−)-calanolide B (**14**) are potent HIV-1 inhibitors, whilst (−)-calanolide A and (+)-calanolide B (**4**) are inactive against the virus [52]. It should be mentioned here that (+)-calanolide A (**1**) is the natural product, but its enantiomer (−)-calanolide A was prepared from the naturally occurring (−)-costatolide (**14**), isolated from *C. costatum*. Similarly, to establish structure-activity-relationships of calanolides, several analogs of calanolides have been synthesized to date, and tested in anti-HIV assays [53]. Although the synthesis of calanolides and the anti-HIV activity of synthetic calanolide analogs are not within the scope of this review, a few examples are given here in the context of structure-activity-relationships. One of the first attempts in this area was from Galinis et al. [53], where ∆ 7,8 olefinic bonds within (+)-calanolide A (**1**) and (−)-calanolide B (**14**) were reduced, and C-12 hydroxyl group in (−)-calanolide B (**14**) was modified to investigate variations in anti-HIV activity compared to parent calanolides. In this study, none of the 14 derivatives was found to possess superior activity to parent calanolides but revealed some preliminary structure-activity requirements for anti-HIV potencies. Later, in order to identify the structural features of naturally occurring (+)-calanolide A (**1**) necessary for its anti-HIV activity and to prepare synthetic analogues, oxo-derivatives (+)-, (−)- and (±)-12-oxocalanolides, were synthesized and tested in vitro using a biochemical reverse transcriptase inhibition assay for determining anti-HIV activity with a promising outcome [48]. In a review article covering various aspects of anti-HIV 4-substitued coumarins with an alkyl or a phenyl group as the substituent, isolated from the genus *Calophyllum*, summarized that all *trans* configurations (10*R*, 11*S*, 12 *S*), as in (+)-calanolide A (**1**) and (+)-inophyllum B (a 4-phenyl-substituted pyranocoumarin), are essential for the best anti-HIV activity [54].

Most of the SAR studies involving calanolides for their anti-HIV activities concentrated on the three chiral centers at C-10, C-11 and C-12 of (+)-calanolide A (**1**) [55,56]. As the number of naturally occurring calanolides are rather limited (calanolides A–F) (Figure 2), the SAR studies were often carried out with natural calanolides as well as their synthetic analogs. Of the diastereomers, compounds containing 10,11¬-*trans*-methylation and 12-(*S*)-OH chirality (Figure 2) displayed the most potent activity with EC50 values in between 0.18 and 2.0 µM [55]. It was also observed that either the enantiomers (12-*R*-OH) or epimeric alcohols, e.g., calanolide C (**6**) could not produce any noticeable anti-HIV effect. It could be concluded that the relative stereochemistry at C-10 and C-11 are essential structural features for potent anti-HIV activity of calanolides, and at the same time, the *S* configuration at C-12 as well as the presence of a heteroatom, e.g., O, at C-12 are necessary for anti-HIV effects.

In order to assess the importance of the presence of 11-methyl functionality on calanolide A for its anti-HIV activity, the activity of the semi-synthetic racemic mixture of 11-demethyl-calanolide A was compared with the anti-HIV activity of its parent compound, (±)-calanolide A [57]. The in vitro HIV-1 reverse transcriptase inhibitory activity of these compounds was determined with the isotope 3H assay, which is a thymidine incorporation assay that often utilizes a strategy wherein a radioactive nucleoside, 3H-thymidine, is incorporated into new strands of chromosomal DNA during mitotic cell division; a scintillation beta-counter is used to measure the radioactivity in DNA recovered from the cells in order to determine the extent of cell division that has occurred in response to a test agent. The cytotoxicity and inhibition of cytopathic effect of (±)-calanolide A and (±)-11-demethyl-calanolide A were studied in HIV-1 IIIB infected MT-4 cell cultures by the MTT staining method. Both compounds inhibited HIV-1 reverse transcriptase in vitro with IC50 value of 3.028 µM/L and 3.965 µM/L, respectively, for (±)-11-demethyl-calanolide and (±)-calanolide A. They also inhibited cytopathic effect in HIV-1 IIIB infected MT-4 cell cultures with IC50 values of 1.081 and 1.297 µM/L, respectively. The outcome form this study indicated that (±)-11-demethyl-calanolide had a slightly more potent anti-HIV activity than (±)-calanolide A, suggesting the methyl functionality at C-11 in calanolide A (**1**) might not be an essential structural feature for anti-HIV activity. With the help of synthetic analogues a few other structural features that could impact on the anti-HIV activity of calanolides could be identified. Some of those are summarized below:

With the advent of various modern computational tools and mathematical models, it is now possible to study quantitative structure activity relationships (QSAR) in silico, and to predict the potential of any drug candidates for any therapeutic application [58]. A Caco-2 cell permeability QSAR model has recently been used to study various HIV-1 reverse transcriptase inhibitors, including (+)-calanolides A (**1**) and B (**4**), both of which showed a high degree of permeability [59]. This parallel computational screening method incorporated approaches of intestinal absorption prediction, receptor affinity estimation, inhibitor shape similarity, lipophilicity, and index-based lipophilic efficiency analyses. Calanolide A (**1**), among a few other HIV-1 reverse transcriptase inhibitors, emerged as one of the prioritized hits, as a result of guided prioritization task by the better binding affinity, crystal ligand similarity, permissible log*P* value and top lipophilic ligand efficiency scores.

#### 4.2.4. Mechanism of Action

The evaluation of the activity of (+)-calanolide A (**1**) against reverse transcriptase and nonnucleoside reverse transcriptase inhibitor-resistant viruses and enzyme kinetic studies for reverse transcriptase inhibition suggest that this coumarin possibly interacts with the HIV-1 reverse transcriptase in a fashion mechanistically different from other known NNTRIs. The biochemical mechanism of inhibition of HIV-1 reverse transcriptases by calanolide A (**1**) was studied using two primer systems, ribosomal RNA and homopolymeric rA-dT(12-18) [60]. Calanolide A (**1**) was found to bind near the active site of the enzyme and interfered with dNTP binding; it inhibited HIV-1 reverse transcriptase in a synergistic fashion with nevirapine, further distinguishing it from the general class of NNRTIs. It was also observed that at certain concentrations, this compound could bind HIV-1 reverse transcriptase in a mutually exclusive manner with respect to both the pyrophosphate analog, phosphonoformic acid and the acyclic nucleoside analogue 1-ethoxymethyl-5-ethyl-6-phenylthio-2-thiouracil. It was concluded that calanolide A (**1**) could share some binding domains with both phosphonoformic acid and 1-ethoxymethyl-5-ethyl-6-phenylthio-2-thiouracil. It might interact with reverse transcriptase near both the pyrophosphate binding site and the active site of the enzyme. Later, the same group of researchers studied possible mechanism of action of action of calanolide A (**1**) against the HIV type 1 including a variety of laboratory strains, with EC50 values of 0.10–0.17 µM [60]. Calanolide (**1**) could inhibit promonocytotropic and lymphocytotropic isolates from patients in various stages of HIV disease, and drug-resistant strains, and was found to act early in the infection process like the known HIV reverse transcriptase inhibitor 2′ ,3′ -dideoxycytidine. It could selectively inhibit recombinant HIV type 1 reverse transcriptase but not cellular DNA polymerases or HIV type 2 reverse transcriptase. Auwerx et al. [50] studied the possible role of Thr139 in the HIV-1 reverse transcriptase sensitivity to (+)-calanolide A (**1**). As T139I reverse transcriptase proved to be resistant to (+)-calanolide A (**1**), represents a catalytically efficient enzyme, and requires only a single transition point mutation (ACA→ATA) in codon 139 could provide some explanation as to why mutant T139I reverse transcriptase virus strains, but not the other strains containing other amino acid changes at this position, predominantly emerge in cell cultures under (+)-calanolide A (**1**) pressure.

Calanolides are non-nucleoside reverse transcriptase inhibitors and mediate their inhibitory effect in two different template primer systems: primed ribosomal RNA template, and homopolymeric poly rA-oligoT12-18 primer. Calanolide A (**1**) was found to inhibit reverse transcriptase by involving two binding sites, and the action is because of the bi-bi ordered mechanism of reverse transcriptase, requiring primer binding prior to polymerization [55]. Calanolide A (**1**) can bind HIV-1 reverse transcriptase in a mutually exclusive manner with the pyrophosphate analogues phosphoformic acid or 1-ethoxymethyl-5-ethyl-6-phenylthio-2-thiouracil. This indicates that calanolide A (**1**) can interact with reverse transcriptase near the pyrophosphate binding site as well as the active site. Unlike general non-nucleoside reverse transcriptase inhibitors, calanolide A (**1**) appears to be at least partially competitive inhibitor of dNTP binding. Clinical and laboratory assessment on viral load and CD4 count indicated that antiviral effects of calanolide A (**1**) appeared to be dose-dependent and maximized on day 14 or 16. Viral life-cycle studies indicated that calanolide A (**1**) could act early in the infection process, similar to the known HIV reverse transcriptase inhibitor 2′ ,3′ -dideoxycytidine. In enzyme inhibition assays, calanolide A (**1**) could potently and selectively inhibit recombinant HIV type 1 reverse transcriptase but not cellular DNA polymerases or HIV type 2 reverse transcriptase within the concentration range tested.

#### *4.3. Antimycobacterial Activity*

The antibacterial (against *Bacillus cereus, B. pumilius, B. subtilis*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Salmonella typhi*, *Staphylocossus aureus and Vibrio cholerae*) and antifungal (against *Alternaria tenuissima*, *Aspergillus fumigatus*, *Aspergillus niger*, *Candida albicans and Candida tropicalis*) properties of *Calophyllum* species and their bioactive secondary metabolites, including calanolides, are already known [6,15,61–67]. Kudera et al. [66] reported in vitro growth inhibitory activity of *C. inophyllum* extract against diarrhea-causing microorganisms, e.g.,*Clostridium difficile infant, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes* and *Salmonella enterica*. The extract was particularly active against *C. perfringens* and *L. monocytogenes* (MIC = 128 µg/mL). Later, calanolide E (**10**) was isolated from *C. wallichianum* and tested for its anti-*Bacillus* activity against *Bacillus cereus, B. megaterium, B. pumilus* and *B. subtilis* [20]. However, calanolide E (**10**) was not bactericidal on the tested Bacillus species, and at the tested concentration.

Based on the initial findings on promising antimicrobial properties of calanolides and *Calophyllum* extracts, efforts have recently been directed to the study on the effect of these compounds on the acid-fast bacillus *Mycobacterium tuberculosis*that causes tuberculosis [17,68,69]. As over the years several antibiotic resistant and multidrug-resistant *M. tuberculosis* strains have emerged, and complicated the existing treatment modalities for tuberculosis, and there has been a recent increase in incidents of tuberculosis globally observed, the need for new effective, safe and affordable antimycobacterial drugs has become paramount. *Calophyllum brasiliense* extract was reported to be active against *M. tuberculosis* (IC50 3.02–3.64 µg/mL), and a follow up HPLC analysis of the active extract provided evidence of presence of calanolides and the antimycobacterial activity induced by *C. brasilliense* was attributed mainly to calanolides A (**1**) and B (**4**) [17]. Earlier, Xu et al. [68] demonstrated that calanolide A (**1**), from Colombian *C. lanigerum*, was active against both drug-susceptible and drug-resistant strains of *Mycobacterium tuberculosis,* e.g., H37Ra (ATCC 25177), H37Rv (ATCC 27294), CSU 19, CSU 33, H37Rv-INH-R (ATCC 35822), CSU 36, CSU 38 and H37Rv-EMB-R (ATCC 35837). Efficacy evaluations in macrophages established that this pyranocoumarin could inhibit intracellular replication of *M. tuberculosis* at concentrations below the minimum inhibitory concentration (MIC) determined in vitro. It was postulated that calanolide A (**1**), like the antitubercular drug rifampicin, could rapidly inhibit RNA and DNA synthesis followed by an inhibition of protein synthesis, and could lead to the generation of a new class of pyranocoumarin-based antitubercular drugs. In this study, the natural calanolides A (**1**), B (**4**) and D (**7**), as well as their semisynthetic analogues were tested, and (+)-calanolide A (**1**) and the semisynthetic analogue, 7,8-dihydrocalanolide B emerged as most effective against tuberculosis with the MIC value of 3.13 µg/mL. While (–)-calanolide B (**14**) was moderately effective, calanolide D (**7**) was found inactive at the highest tested concentration of 12.5 µg/mL. In fact, calanolides, especially calanolide A (**1**), is unique in a sense that these compounds have anti-HIV property and were found to be active against *M. tuberculosis* (MIC = 3.1 µg/mL) and an array of drug-resistant strains (MIC = 8–16 µg/mL). The antimycobacterial activity of calanolide A (**1**) is comparable to that of the well-known anti-tubercular drug isoniazid, and effective against rifampicin- and streptomycin-resistant *M. tuberculosis* strains. A recent patent described potent antimycobacterial property of calanolides and their analogs and provided a method of using these compounds for the treatment and prevention of mycobacterial infections [70].

#### *4.4. Antiparasitic Activity*

Traditionally, natural products, especially in crude forms, have long been used to treat various parasitic diseases, like babesiosis, leishmaniasis, malaria, trypanosomiasis and so on. Recently, leishmaniasis and trypanosomiasis have been in research focus of natural products researchers, aiming at discovering new drug candidates to treat these neglected diseases [71,72]. Extracts of *C. brasiliense* and *C. inophyllum* and calanolides were shown effective against intracellular parasites causing American trypanosomiasis and leishmaniasis [6]. In a recent study, Silva et al. (2020) [14] demonstrated that the MeOH extract from stem bark of *C. brasiliense* was active against amastigote forms of *Trypanosoma cruzi* and *Leishmania infantum*. Bioactivity-guided purification of the extra afforded calanolides E1 (**11**) and E2 (**12**), which were found to be active against *T. cruzi* (EC50 values of 12.1 and 8.2 µM, respectively) and *L. infantum*, (EC50 values of 37.1 and 29.1 µM, respectively) in vitro. Calanolide E1 (**11**) displayed the best selectivity index (SI) with values >24.4 to *T. cruzi* and >6.9 to *L. infantum* in comparison to calanolide E2 (**12**). It was concluded that these coumarins could be utilized as scaffolds for the design and development of novel drug candidates to treat Leishmaniasis and Chagas diseases.

#### **5. Toxicological Aspects Including Pharmacokinetics**

Among the naturally occurring calanolides (Figure 2), calanolide A (**1**), a specific nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, first isolated from a tropical tree *C. lanigerum* that grows abundantly in the Malaysian rain forest, is the most-studied compound in terms of its pharmacology, toxicology and synthesis. A series of animal studies [43] involving mice, rats and dogs established that calanolide A (**1**) is generally well-tolerated at oral doses of up to 150 mg/kg in rats and 100 mg/kg in dogs, and possesses a good safety profile [73,74]. Calenolides A (**1**), B (**4**) and C (**6**) were found to be nontoxic in mice (LD50 = 1.99 g/kg), and no alternation on hepatocytes could be observed during the histological study of the mice treated with the highest dose applied [74]. During a study looking at the anti-HIV efficacy and toxicity of calanolides when used in combination with other anti-HIV drugs, no noticeable toxicity could be detected [46].

In the very first study on the safety and pharmacokinetics of calanolide A (**1**) in healthy HIV-negative human volunteers revealed that the toxicity of calanolide A (**1**) was minimal in the majority of subjects treated with four successive single dose, 200, 400, 600 and 800 mg. While there were no acute serious or life-threatening adverse effects were observed, among the usual minor adverse effects, dizziness, oily taste, headache, eructation, and nausea were noticed, but were of minimal clinical significance. These adverse effects were non-dose-dependent [73]. In this study, it was found that calanolide A (**1**) was rapidly absorbed following administration, with time to maximum concentration of drug in plasma (*T*max) values, depending on the doses, occurring between 2.4 and 5.2 h. It was noted that the levels of calanolide A (**1**) in human plasma were higher than would have been predicted from animal studies, but the safety profile was benign. However, taking calanolide A (**1**) with food was found to generate significant variability in pharmacokinetics, but with no detectable interaction with food. Later, these findings were further confirmed by another similar study carried out by Eiznhamer et al. [75]. Calanolide A (**1**), the first member of the new family of NNRTIs, was found to have long elimination half-life, the benign toxicity profile, to achieve trough plasma levels approximating the EC90 of calanolide A (**1**) for HIV-1, to have the potential for twice daily dosing, and to offer the unique HIV-1 resistance profile could make this compound an attractive candidate for further clinical studies. It was reported that after oral administration, (+)-calanolide A (**1**) was generally well tolerated and indication of any safety concern could be observed [48]. Its plasma concentrations in humans were higher than anticipated from animal data. The AUC and Cmax values increased with increasing dose, and it appeared that therapeutic levels could easily be achieved in humans.

A comparative study on the relative pharamacokinetic parameters and bioavailability of calanolide A (**1**) and its synthetic analogue dihydrocalanolide A (**15**) was reported [76]. This study compared the intravenous pharmacokinetics of the dihydro analog relative to the parent compound, calanolide A (**1**), and determined the relative oral bioavailability of each drug in CD2F1 mice. Both compounds displayed similar pharmacokinetic parameters, but the oral bioavailability of the dihydro analogue was considerably better (almost 3.5-fold) than calanolide A (**1**). Moreover, the relative ability of calanolide A (**1**) and its dihydro analog to change to their inactive epimer forms, (+)-calanolide B (**4**) and (+)-dihydrocalanolide B, respectively, was also determined; while conversion of active calanolides to inactive forms occurred in vitro especially under acidic conditions, no epimers of either compound were observed in plasma of mice after administration of either (+)-calanolide A (**1**) or (+)-dihydrocalanolide A (**15**). It was suggested that the selection of the dihydro derivative of calanolide A (**1**) could be a reasonable choice for further preclinical development and possible Phase I clinical evaluation as an oral drug candidate for the treatment of HIV infection. Calanolide A (**1**) was shown to be distributed readily into the brain and lymph [55]. The distribution and elimination pattern of calanolide A (**1**) and its 7,8-dihydro derivative were found to be similar, but the apparent volume of distribution (Vd) and oral clearance of these compounds were significantly different after oral administration. It was also demonstrated that calanolide A (**1**) is generally well tolerated in doses up to 600 mg. As evident from animal studies, the gastrointestinal intolerance for this compound is not severe, but the most common adverse events as observed in human trails of calanolide A (**1**) include an oily after taste and transient dizziness [55]. The calculated half-life of calanolide A (**1**) from 800 mg dosing was reported to be 20 h [55,73].

During the study directed to the evaluation of antitubercular property of calanolides and their semisynthetic analogues, the pharmacokinetic data indicated that the (+)-calanolide A (**1**) concentrations in plasma could be comparable to the observed in vitro MICs against *M. tuberculosis* [68]. Both calanolides A (**1**) and B (**4**) metabolized by cytochrome P450 CYP3A, and their blood levels could be enhanced if co-administered with ritonavir. Usach et al. [77] reported the safety, tolerability and pharmacokinetics profiles of calanolide A (**1**), as a result of a comprehensive Phase I clinical trial.

#### **6. Therapeutic Potential**

Naturally occurring calanolides and their synthetic or semi-synthetic analogs have undergone several pre-clinical and clinical trials for their anti-HIV activity, aiming at novel anti-HIV drug development [2,16,55,78]. In fact, calanolide A (**1**) was at an advanced stage of development as an anti-HIV drug about a decade ago [78]. Buckheit [79] reviewed therapeutic potential of non-nucleoside reverse transcriptase inhibitors like calanolides as anti-HIV and commented on strategies for the treatment modalities for HIV infections. In fact, NNRTIs opened a new avenue of treatment of HIV infections, as previously this therapeutic area was predominantly covered by nucleoside reverse transcriptase inhibitors and protease inhibitors. Soon after the discovery of calanolides as a potential ant-HIV agents by the NCI/NIH, Sarawak Medichem Pharmaceuticals (Sarawak, Malaysia) synthesized calanolide A (**1**) and started developing calanolide A (**1**) as a clinical drug for the treatment of HIV infections. It was a joint venture between the Sarawak State Government and Medichem Research Inc.

During 2001–2005, an interventional clinical trial was conducted on human volunteers [80], where patients were randomized to receive (+)-calanolide A (**1**) or placebo for 21 days. All patients could elect to receive an open-label, 3-month course of approved retroviral therapy (up to triple-drug therapy) to be selected by, and administered under the care of, the patients' physicians. If the patient had no insurance coverage or did not wish to utilize his/her insurance for anti-HIV medications, Sarawak MediChem Pharmaceuticals provided these medications at no charge for up to three months. The trial was primarily aimed at the assessment of the safety and effectiveness of (+)-calanolide A (**1**) in HIV-infected patients who had never taken anti-HIV drugs. In 2006, Craun Research (Kuching, Malaysia), a company established by the Sarawak Government, acquired Sarawak MediChem, and in 2016, Craun Research announced the completion of Phase I clinical trials for calanolide A (**1**) with doses of 200 to 800 mg, which initially started in 2013 [77]. In 2017, F18 (10-chloromethyl-11-demethyl-12-oxo-calanolide A), a synthetic structural analog of calanolide A (**1**) was shown to have more potent anti-HIV activity than original molecule, calanolide A (**1**) [81,82]. This compound showed better druggable profile with 32.7% oral bioavailability in rat, tolerable oral single-dose toxicity in mice, and suppressed both the wild type HIV-1 and Y181C mutant HIV-1 at an EC50 of 7.4 nM and 0.46 nM, respectively [83]. Furthermore, it was shown that two enantiomers F18, (*R*)-F18 and (*S*)-F18, had quite similar anti-HIV property, but (*R*)-F18 was more potent than (*S*)-F18 against wild type virus, K101E mutation and P225H mutation pseudoviruses [81]. However, calanolides, particularly calanolide A (**1**) remains as an investigational anti-HIV drug and has not yet been approved by the FDA or any other drugs regulatory bodies for their commercial pharmaceutical production.

#### **7. Patents**

In 1999, calanolides and related antiviral compounds were patented by the Board of Trustees of the University of Illinois [84]. The patent covered novel antiviral compounds, calanolides, and their derivatives that could be isolated from plants of the genus *Calophyllum* in accordance with the specified method. The patent also included the uses of these compounds and their derivatives alone or in combination with other antiviral agents in compositions, such as pharmaceutical compositions, to inhibit the growth or replication of a virus, such as a retrovirus, in particular a human immunodeficiency virus, specifically HIV-1 or HIV-2. Later, another patent, owned by Parker Hughes Institute, was reported, which described the novel uses of calanolides as Tec family/BTK (Bruton's tyrosine kinase) inhibitors, methods for their identification, and pharmaceutical compositions [85]. It can be mentioned here that the BTK inhibitors inhibit the enzyme BTK, which is a crucial part of the B-cell receptor signaling

pathway, and these inhibitors have emerged as a new therapeutic target in a variety of malignancies, e.g., chronic lymphocytic leukemia and small lymphocytic lymphoma [86].

#### **8. Conclusions**

Non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz, nevirapine and delavirdine, have become one of the cornerstones of highly active anti-retroviral therapy for HIV infections. Calanolides, as they belong to this pharmacological class of NNRTIs, and because of their high safety margins and favorable pharmacokinetic profiles, are ideal candidates for novel anti-HIV drug development. While several analogues of the naturally occurring calanolides have been synthesized, a good number of preclinical and clinical trials have been conducted to date, and there are a few patents published, further work is still required to commercially bring any of the calanolide candidates, natural or synthetic, to anti-HIV drug market. As calanolides show an excellent synergistic and additive profile in combination with other anti-HIV drugs, it is assumed that calanolides can be considered for use in combination therapy for HIV infections.

**Author Contributions:** All authors contributed equally to the data collection and compilation of information. Additionally, L.N. and S.D.S. played the lead role in writing, editing and submission of this manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the European Regional Development Fund—Project ENOCH (No. CZ. 02.1.01/0.0/0.0/16\_019/0000868).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Chalepin and Chalepensin: Occurrence, Biosynthesis and Therapeutic Potential**

**Lutfun Nahar 1,\* , Shaymaa Al-Majmaie 2 , Afaf Al-Groshi 2 , Azhar Rasul 3 and Satyajit D. Sarker 2,\***

**Abstract:** Dihydrofuranocoumarin, chalepin (**1**) and furanocoumarin, chalepensin (**2**) are 3-prenylated bioactive coumarins, first isolated from the well-known medicinal plant *Ruta chalepensis* L. (Fam: Rutaceae) but also distributed in various species of the genera *Boenminghausenia*, *Clausena* and *Ruta*. The distribution of these compounds appears to be restricted to the plants of the family Rutaceae. To date, there have been a considerable number of bioactivity studies performed on coumarins **1** and **2**, which include their anticancer, antidiabetic, antifertility, antimicrobial, antiplatelet aggregation, antiprotozoal, antiviral and calcium antagonistic properties. This review article presents a critical appraisal of publications on bioactivity of these 3-prenylated coumarins in the light of their feasibility as novel therapeutic agents and investigate their natural distribution in the plant kingdom, as well as a plausible biosynthetic route.

**Keywords:** *Ruta chalepensis*; Rutaceae; chalepin; chalepensin; bioactivity; biosynthesis

#### **1. Introduction**

Chalepin (**1**; mol formula: C19H22O4; mol weight 314) and chalepensin (**2**; mol formula: C16H14O3; mol weight 254) (Figure 1) are, respectively, a dihydrofuranocoumarin and a furanocoumarin, with a prenylation at C-3 of the coumarin core structure. These coumarins, as the names imply, were first isolated from *Ruta chalepensis* L. (Fam: Rutaceae), but are also found in other *Ruta* species, e.g., *R. angustifolia* and a few other plants of the genus *Clausena* (Fam: Rutaceae), e.g., *Clausena anisata* (Willd.) Hook. F. ex Benth. [1–4]. While chalepin (**1**), also known as heliettin, is optically active, chalepensin (**2**), also known as xylotenin, does not possess any optical activity. Although these coumarins are rather rare in the sense that there are not many 3-prenylated naturally occurring furanocoumarins reported to date, there are quite a good number of bioactivity studies carried out on these compounds. The present review critically appraises publications on bioactivity of these 3-prenylated furanocoumarins in the light of their feasibility as novel therapeutic agents and covers their natural distribution in the plant kingdom, as well as a plausible biosynthetic route.

−

−

−

−

−

− −

− −

**Citation:** Nahar, L.; Al-Majmaie, S.; Al-Groshi, A.; Rasul, A.; Sarker, S.D. Chalepin and Chalepensin: Occurrence, Biosynthesis and Therapeutic Potential. *Molecules* **2021**, *26*, 1609. https://doi.org/10.3390/ molecules26061609

Academic Editor: Maria João Matos

Received: 24 February 2021 Accepted: 12 March 2021 Published: 14 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Distribution**

First isolated from *Ruta chalepensis* more than half a century ago, chalepin (**1**) and chalepensin (**2**) have been further reported mainly from various species of the genera *Clausena* and *Ruta* of the family Rutaceae [4,5]. It appears that these compounds exclusively occur in the family Rutaceae [1–20], and predominantly within these two genera. However, *Boenminghausenia albiflora* var. *japonica (Hook.)* Rchb. Ex Meisn and *B. sessilicarpa* H. Lev. also produce chalepensin (**2**) [6,20] and this genus is phylogenetically close to the genus *Ruta* [21]. Chalepensin (**2**) was further found in the leaves of *Esenbeckia alata* (Karst and Triana) Tr. and Pl. [9], while *E. grandiflora* Mart. was reported to produce chalepin (**1**) [10]. Interestingly, the genus *Esenbeckia* Kunth. is a part of a small group of phylogenetically distant Rutaceae including the genera *Clausena* and *Ruta,* where 3-prenylated coumarins like **1** and **2** are generally produced [9]. Thus, co-occurrence of these 3-prenylated furanocoumarins in these genera might have some chemotaxonomic implications, at least at the family level, within the family Rutaceae. The distribution of these two coumarins (**1** and **2**) is summarized in Table 1. Within the source plants these compounds are well distributed almost in all parts, leaves, stem, flowers and fruits. Although not chalepensin (**2**) itself, a series of 5-*O*-prenylated chalepensin derivatives were reported from *Dorstenia foetida* Schweinf., a medicinal plant from the family Moraceae, distributed in various countries in the Middle-East Asia [22].

**Table 1.** Distribution of chalepin (**1**) and chalepensin (**2**) in the plant kingdom.

+ = Found; − = Not found.

#### **3. Biosynthesis**

Like all other coumarins, the biosynthesis of chalepin (**1**) and chalepensin (**2**) begins from the simple coumarin umbelliferone, which is formed from the amino acid L-phenylalanine through the formation of *trans*-cinnamic acid, *p*-coumaric acid, 2-hydroxy*p*-coumaric acid, 2-glucosyloxy-*p*-coumaric acid and 2-glucosyloxy-*p*-*cis*-coumaric acid aided by different enzymes, e.g., cinnamate 4-hydroxylase and 4-coumarate-CoA ligase, 4-coumaroyl 2′ -hydroxylase (Figure 2) [23,24]. Sharma et al. [25] studied the biosynthesis of chalepin (**1**) in *Ruta graveolens*. They suggested that 3-(1,1-dimethylallyl)-umbelliferone could be the key intermediate for the biosynthesis of chalepin (**1**), and the dihydrofuran moiety in chalepin (**1**) is formed via prenylation, aided by dimethylallyldiphosphate, at C-6 of the core coumarin skeleton followed by oxidative cyclization with neighboring hydroxyl function at C-7. Generally, prenyltransferases (6-prenyltransferase was identified in *R. graveolens* as a plastidic enzyme) are considered the enzymes involved in the biosynthesis of furano-/dihydrofuranocoumarins through umbelliferone prenylation. Further oxidation of chalepin (**1**) could lead to the formation of the furanocoumarin chalepensin (**2**) in a similar fashion as observed in the conversion of marmesin to psoralen [26]. In fact, biosynthesis of chalepin (**1**) resembles that of 3-prenylated furanocoumarin, rutamarin

(acetyl-chalepin) [26]. At this moment, it is not clear from the literature if the prenylation at C-3 takes precedence over that on C-6. In fact, the published information on the biosynthesis of these coumarins **1** and **2** is rather extremely limited, and much work, especially using radioisotopes is much needed to explore other possible routes to the biosynthesis of these compounds.

### A = 3-Prenylation; B = 6-Prenylation; C = Oxidative cyclization; D = Oxidation

**Figure 2.** Putative biosynthetic route for the formation of chalepin (**1**) and chalepensin (**2**).

#### **4. Bioactivity**

′

The general, *Clausena* and *Ruta*, the main sources of chalepin (**2**) and chalepensin (**2**), are well known for their uses in traditional medicines, and different studies have established their bioactivities [27,28]. Chalepin (**1**) and chalepensin (**2**) have emerged as two major bioactive components in many of those plants through bioassay-guided isolation protocols, and their bioactivities include antimicrobial, anti-inflammatory, anticancer, antiviral and many more. In this section, using several subsections, a critical appraisal is presented on bioactivities of these two compounds (**1** and **2**) reported in the literature to date (Table 2) [29–51]. Most of the reported bioactivity studies on these compounds involved predominantly in vitro assays and only a handful of in vivo and in silico studies. However, there is no report on any systematic preclinical or clinical trial with these compounds involving human volunteers available in the literature to date.

NR = No report available; + = Active.

#### *4.1. Antidiabetic Activity*

Among the bioactive compounds isolated from the stem bark of *Clausena lansium* (Lour.) Skeels, chalepin (**1**) exhibited antidiabetic properties, exerted through dose-dependent stimulated (glucose-mediated) insulin release in vitro from INS-1 cells (rat insulinoma cell line) [8]. Chalepin (**1**) showed 138% insulin secretory response in vitro at the concentration of 0.1 mg/mL. INS-1 cells are widely used as rat islet β-cell models for screening for antidiabetic properties of plant extracts or purified compounds. They express muscarinic M1 and M3 receptors, which are activated by carbachol to promote insulin release. Chalepensin (**2**) does not appear to have gone through any antidiabetic screening yet. It is known that insulin secretion involves a sequence of events in β-cells that lead to fusion of secretory granules with the plasma membrane; it is secreted primarily in response to glucose, while other nutrients such as free fatty acids and amino acids can augment glucose-induced insulin secretion.

#### *4.2. Antifertility Activity*

During the assessment of the extracts of *R. chalepensis* var *latifolia* for antifertility activity in rodents, chalepin (**1**) and chalepensin (**2**) were discovered as the major active antifertility principles in the extracts [13]. Despite these compounds showing antifertility activity, most of the tested animals developed cystic and atretic follicles in their ovaries and glomerulocapsular adhesion and segmental fusion in the kidneys. However, no brain toxicity was observed with these compounds. Kong et al. [29] assessed the antifertility activity of the chloroform extracts of the roots, stem and leaves of *R. graveolens* L. in rats and fractionation of the extracts afforded coumarin **2** as the active component with moderate toxicity. Time-dosing experiments showed that this coumarin (**2**) could act at the early stages of pregnancy. The observed antifertility activity of **1** and **2** [13,29] could provide some scientific evidence in support of the traditional uses of *R. chalepensis* as an abortifacient.

#### *4.3. Antimicrobial Property*

Antimicrobial assay-guided analysis of a root extract of *Clausena anisata* (Willd.) Hook. f. Benth., a well-known medicinal plant used traditionally for the treatment of parasitic infections, influenza, abdominal pain and constipation, afforded chalepin (**1**) as an antibacterial agent, particularly effective against *Bacillus subtilis* with a zone of inhibition of 16 mm as opposed to 15 mm of the positive control cifrofloxacin [4]. This coumarin was also found active against two other pathogenic bacterial strains, *Staphylococcus aureus* and *Pseudomonas aeruginosa*. Chalepensin (**2**), on the other hand, was reported to possess antifungal property and was found to inhibit the growth of the fungal strains, *Candida albicans* and *Cryptococcus neoformans* [30]. However, interestingly, none of these coumarins showed any antimicrobial activity at tested concentrations (50-100 µg/mL) against a range of microorganisms, e.g., *Bacillus subtilis*, *Mycobacterium smegmatis*, *Staphylococcus aureus* and *Candida albicans*, using a modified microtitre-plate assay as reported by El Sayed et al. [52]. Chalepensin (**2**), isolated from *R. chalepensis*, was assessed for antibacterial activity against *Streptococcus mutans* using the method of colony forming units counts in solid medium culture and reduction of tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in liquid medium [31] and was shown to significantly inhibit the growth of this bacterial strain with an MIC (minimum inhibitory concentration) of 7.8 µg/mL.

In the most recent study [32] on anti-MRSA (methicillin-resistant *Staphylococcus aureus*) activity of several compounds, mainly coumarins and flavonoids, isolated from *R. chalepensis* grown in Iraq, both chalepin (**2**) and chalepensin (**3**) showed significant antimicrobial activities against the MRSA strains, ATCC 25923, SA-1199B, XU212, MRSA-274819 and EMRSA-15 with MIC values ranging between 32 and 128 µg/mL. In that study, two other furanocoumarins, bergapten and isopimpineline, which do not have a 3-prenylation as in **1** and **2**, were found inactive at tested concentrations. Based on this finding, it was suggested that the prenylation at C-3 of the coumarin nucleus might be a key determi-

nant of anti-MRSA activity. Chalepensin (**2**) was found to be more active than chalepin (**1**) and was subjected to in silico studies to gain an insight into the extent at which this compound (**2**) is able to bind to MRSA proteins and also their drug−like physicochemical characters. In silico studies on compound **2** showed that this compound could have high GI absorption and no violation of the Lipinski rules. It was also shown that chalepensin (**2**) could bind with certain MRSA protein targets, predominantly through hydrogen bonding as well as van de Waals forces. It was suggested that this coumarin could be utilized as a structural template for generating structural analogs and developing potential anti-MRSA therapeutic agents.

#### *4.4. Antiprotozoal Activity*

One of the major traditional medicinal uses of *R. chalepensis* and other *Ruta* species is their efficacy as antiparasitic agents [28], particularly as an anthelmintic medication. This traditional medicinal use of *R. chalepensis* has prompted antiparasitic activity screening of its extracts and isolated major compounds, including chalepin (**1**) and chalepensin (**2**). Antiprotozoal activity of chalepin (**1**) and chalepensin (**2**), obtained from *R. chalepensis* following a bioassay-guided protocol, against *Entamoeba histolytica*, which is a causative organism of ameoebiasis, was reported as a meeting presentation, but no further full scientific report was published [33]. Both coumarins showed >90% growth inhibition against *E. histolytica*, an anaerobic parasitic amoebozoan, at a concentration of 150 µg/mL with IC50 values of 28.67 and 38.71 µg/mL, respectively, for compounds **1** and **2**. However, in a previous study [5], conducted by the same group, evaluated antiprotozoal activity of plants used in northwest Mexican traditional medicine, particularly *Lippia graveolens* Kunth. and *R. chalepensis*, against *E. histolytica,* and chalepensin (**2**) was found to be the main antiprotozoal component in *R. chalepensis*. Earlier, Kundu and Roy [34] carried out in silico studies involving chalepin (**1**) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the pathogenic protozoa *E. histolytica.* It can be noted that GAPDH is a major glycolytic enzyme (~37 kDA), which catalyzes the sixth step of glycolysis, and an attractive drug target like *E. histolytica* lacks a functional citric acid cycle and exclusively depends on glycolysis for its energy needs. Chalepin (**1**) was predicted as a GAPDH inhibitor and structural modifications offering additional polar interactions were suggested to improve potency.

*Trypanosoma cruzi*, a species of parasitic euglenoids, characteristically can bore tissue in another organism and feed on blood and lymph, causing diseases like Chagas disease (also known as American trypanosomiasis) in humans, that affects more than 7 million people worldwide, with Latin American countries being most affected. In recent years, a renewed interest has been observed in the search for antitrypanosomal natural products, especially from higher plants. In many in vitro as well as in silico studies, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) from *T. cruzi* has been used as a target molecule for screening compounds for potential antitrypanosomal activity [35]. In an in silico study with various natural products, chalepin (**1**) emerged as a hit molecule for antitrypanosomal drug discovery [36], and subsequently, a series of 3-piperonylcoumarins were synthesized and tested for their inhibitory activity against gGAPDH. Chalepin (**1**) was shown in silico to possess the highest binding affinity to gGAPDH (IC50 = 55.5 µM) among the natural coumarins screened and the best inhibitor of gGAPDH [36,37]. Earlier, during an in vitro screening of natural coumarins for trypanocidal or antitrypanosomal activity, chalepin (**1**) was found to be the most active coumarin with an IC50 value of 64 µM [38]. However, to the best of our knowledge, there is no report available to date on antitrypanosomal property of chalepensin (**2**).

#### *4.5. Antiviral Activity*

Chalepin (**1**), isolated from *R. graveolens*, along with its 28 synthetic analogs were tested for their inhibitory activity on the Epstein–Barr virus (EBV, also known as human herpes virus 4) lytic replication activity [41]. It was noted that most of the synthesized

analogs were more active than their parent or precursor, (-)-chalepin (**1**). EVP is a human gamma-herpes virus that infects more than 90% of the human population globally, and preferentially infects B lymphocytes and epithelial cells causing various diseases like Hodgkin's disease, Burkitt's lymphoma, nasopharyngeal carcinoma and gastric carcinoma in humans. Thus, inhibition of EBV lytic replication is considered as one of the pragmatic strategies for the treatment of some these diseases.

Chalepin (**1**), isolated from the leaves of *R. angustifolia*, displayed significant inhibitory activity (IC50 = 1.7 µg/mL) against hepatitis C virus replication and was found to be more potent than the positive control ribavirin (IC50 = 2.8 µg/mL), a well-known antiviral drug used for the treatment of hepatitis C and other viral diseases [39]. In continuation of their study, they have recently reported enhancement of antihepatitis C virus activity of chalepin (**1**) in combination with conventional antiviral drugs including cyclosporine A, daclatasvir, ribavirin, simeprevir and telaprevir [40]. It was found that chalepin (**1**) could enhance antihepatitis C activities of these conventional drugs with a synergistic combination index of <1. It could be considered as an excellent finding as the need for new and effective drugs for treating hepatitis C is of paramount importance. It can be mentioned that hepatitis C virus infects around 71 million people globally, causes severe liver disease, e.g., liver cancer and deaths; the WHO (World Health Organization) estimated that in 2016, about 400,000 people died from hepatitis C, mainly from liver cirrhosis and liver cancer [53,54].

Like many other antiviral coumarins including some 3-substitued ones, it can be assumed that chalepin (**1**) might offer antiviral activity through inhibition of various proteins that are involved in the transcription/translation processes essential for viral life cycle at different stages, and via modulation of host cell signaling, NF-kB (nuclear factor κB), and inflammatory redox-sensitive pathways and thus blocking viral replication [54]. However, clearly, further research is necessary to understand and establish definite mode of antiviral action mechanism of chalepin (**1**). However, there is no data available on any antiviral property of chalepensin (**2**) to date.

#### *4.6. Cytotoxicity (Potential Anticancer and Antitumor Activity)*

Cancer is one of the major causes of human mortality and morbidity. Currently available cancer treatment options or modalities are rather limited, and often suffer from severe side effects. Therefore, the search for new, effective, safe and affordable anticancer drugs is a part of many major modern drug discovery initiatives worldwide. Natural products have long been considered one of the major contributors in the continuing search for new anticancer molecules for safer and more effective anticancer drug development, and evidently, have already provided several successful anticancer drugs, e.g., taxol, vincristine and vinblastine [55]. The most common starting point in the search for anticancer molecules is the screening compounds for cytotoxicity against various human cancer cell lines because cytotoxicity is regarded as one of the major characteristics of anticancer agents. In order to assess anticancer potential of chalepin (**1**) and chalepensin (**2**), cytotoxicity of these compounds has been assessed against different human cancer cell lines in vitro, and some mechanistic studies on how they kill the cancer cells have also been published, showing anticancer and antitumor potential of these compounds (Table 2).

Chalepin (**1**), isolated from *Clausena emarginata* C. C. Huang, has been found to possess significant cytotoxicity against five human cancer cell lines including human leukemia (HL-60), hepatocarcinoma (SMMC-7721), lung carcinoma (A-549), breast cancer (MCF-7) and colon adenocarcinoma (SW-480) with IC50 values comparable to that of the positive control, doxorubicin [7]. Chalepin (**1**), isolated from *Ruta angustifolia* Pers., was demonstrated to induce apoptosis through phosphatidylserine externalizations and DNA fragmentation in breast cancer cell line, MCF-7 [12,42]; this compound was considerably cytotoxic to MCF-7 cells, moderately cytotoxic to the epithelial human breast cancer cells (MDA-MB231), but not cytotoxic to normal cells, MRC-5 (Medical Research Council cell strain 5) in the SRB (sulforhodamine B) assay [56]. MRC-5 is a diploid cell culture line comprising fibroblasts, first developed from the lung tissue of a 14-week-old aborted Caucasian male fetus. It can be

mentioned here that apoptosis is a process by which cell commit suicide and is eliminated from the system; induction of apoptosis, a cell toxicity pathway, is considered as one of the early-stage mechanism for compounds to exert anticancer activity. This differential cytotoxicity against cancer cells and noncancerous cells might make this compound an ideal candidate, or at least a structural template, for anticancer drug development.

Earlier, in order to understand how chalepin (**1**) could exert its anticancer potential, a study conducted by Richardson et al. [11], revealed that this compound could dosedependently exhibit cell cycle arrest at S phase, suppress nuclear factor kappa B (NFκB) pathway, signal transducer and activation of transcription 3 phosphorylation and extrinsic apoptotic pathway in human non-small cell lung cancer cell line A-549. Cell cycle analysis using the flow cytometry confirmed that chalepin (**1**) could inhibit cell cycle at S phase (synthesis phase), which is the phase of the cell cycle, where DNA is replicated and occurs between the G1 and G2 phases. Since accurate duplication of the genome is essential for successful cell division to take place, the processes involved in the S phase are tightly regulated and widely conserved. A significant accumulation of cells in the S phase was observed after chalepin (**1**) treatment (45 µg/mL) for 48 (accumulation 27.7%) and 72 h (accumulation 25.4%), whereas the accumulation was only about 4% for the untreated cells [11]. It is well known that there is a remarkable link between cell cycle and cancer, as cell cycle appears to be the machinery that controls cell proliferation, and uncontrolled cell proliferation happens in cancer. The suppression of the NF-κB pathway by chalepin (**1**) was shown to be through modulation of the p65 subunit of NF-κB, where the phosphorylation of p65 and the translocation of the p65 subunit to nucleus were inhibited [11]. It can be noted that the NF-κB pathway is generally induced by carcinogens and inflammatory agents. Thus, suppression of NF-κB pathway by chalepin (**1**) could suggest its potential as an anticancer agent.

Caspase 8 is implicated to the activation of the intrinsic apoptotic pathway, and enhancement of caspase 8 activity can be exploited to identify compounds with plausible anticancer activity. In chalepin (**1**) treated cells, a significantly increased level of caspase 8 activity was noticed, when compared to the control; after 48 and 72 h of incubations, chalepin (**1**) (45 µg/mL) enhanced caspase 8 activity, respectively, by 5-fold and 8.6-fold [11].

This group of researchers also demonstrated that chalepin (**1**) and chalepensin (**2**) could induce mitochondrial mediated apoptosis in lung carcinoma cells (A-549), with chalepin (**1**) being more cytotoxic than chalepensin (**2**) [3]; chalepin (**1**) exhibited selective cytotoxicity against A-549 cells with an IC50 value of 8.69 µg/mL (27.64 µM). Chalepin (**1**) was mildly toxic to the normal cell line with an IC50 value of 23.4 µg/mL. Chalepensin (**2**) exhibited considerable cytotoxic property against A-549 cell line with IC50 value of 18.5 µg/mL, while the cytotoxicity (IC50 = 23.4 µg/mL) of this coumarin against noncancerous MRC-5 human lung fibroblast cell line was of moderate level as was with chalepin (**1**). Chalepin (**1**) showed morphological changes, typical for apoptosis, e.g., plasma membrane blebbing, cell vacuolization, echinoid spiking, chromatin condensation, formation of apoptotic bodies, cell shrinkage and nuclear fragmentation. Both coumarins (**1** and **2**) were found to downregulate inhibitors of apoptosis such as Bcl-2, survivin, Bcl-xl and cFLIP. They also triggered release of cytochrome c and activated caspases 9 and 3 to induce apoptosis. Chalepensin (**2**) was shown to possess cytotoxicity against colon (H-T29), lung (A-549), breast (MCF-7), kidney (A-498), and pancreatic (PACA-2) cancer cell lines [3].

Wu et al. [17] screened 19 compounds isolated from *Ruta chalepensis*, including chalepensin (**2**), for their potential cytotoxicity against KB (keratin forming tumor), Hela, DLD (colorectal adenocarcinoma) and Hepa tumor cell lines, but chalepensin (**2**) was found to be inactive against any of these cell lines at tested concentrations. From the available literature data, it is obvious that chalepin (**1**) is more cytotoxic than chalepensin (**2**). However, considerably more work has been carried out with chalepin (**1**) than with chalepensin (**2**) to date, and further comparative work may be necessary to gain a better insight into their anticancer potential.

#### *4.7. Miscellaneous Activities*

Spasmolytic activities of chalepin (**1**) and a few other coumarins, isolated from *Boenninghausenia albiflora* (Hook.) Rchb. Ex Meisn., were reported by Rizvi et al. [43]. Effects of aqueous extracts of *R. graveolens* and its ingredients, chalepensin (**2**) being one of them, on major drug metabolizing enzymes, cytochrome P450, uridine diphosphate (UDP)-glucuronosyltransferase and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-quinone oxidoreductase, were evaluated in mice [15]. The repeated administration of *R. graveolens* extract, rich in rutin and chalepensin (**2**), could induce hepatic CYP1a and CYP2b activities in a dose-dependent fashion. It was observed that male mice were more responsive than female mice to the extract-medicated induction of UGT (uridine glucuronosyltransferase). Earlier, the same group of researchers [48] showed mechanism-based inhibition of CYP1a1 and CYP3A4 by chalepensin (**2**), while this compound was also found to inhibit human CYP1a2, CYP2a13, CYP2c9, CYP2d6 and CYP2e1.

In order to study the in vivo effect of chalepensin (**2**), Lo et al. [44] assessed its effect on multiple hepatic P450 enzymes in C57BL/6JNarl mice, and observed that this coumarin, after oral administration (10 mg/kg) in mice for 7 days, could decrease hepatic coumarin 7-hydroxylation by CYP2a, and increase 7-pentoxyresorufin *O*-dealkylation by CYP2b, without affecting the activities of other CYP enzymes. It was further observed that the suicidal inhibition of CYP2a5 and the constitutive androstane receptor (CAR) mediated CYP2b9/10 induction simultaneously happened in chalepensin (**2**)-treated mice. Previously, Ueng et al. [46,47] and Lo et al. [45] carried out related extensive studies on mechanismbased inhibition of CYP enzymes by chalepensin (**2**) in various in vitro and in vivo models. However, there is no report on such activities of chalepin (**1**) available in the published literature to date.

In a study conducted by Wu et al. [17], chalepensin (**2**) at 100 µg/mL concentration displayed significant antiplatelet aggregation activity, induced by arachidonic acid and collagen. This coumarin, isolated from *Boenninghausenia albiflora* var*. japonica,* was also reported to possess calcium antagonistic property [6].

#### **5. Mutagenicity and Other Toxicities**

The mutagenicity of chalepin (**1**) was assessed at the HGPRT locus (AzGr) in Chinese hamster V79 cells [50], and this compound was found to be mutagenic. Chalepin (**1**), isolated from *Clausena aniseta*, a well-known medicinal plant from West Africa, showed anticoagulant (blood-thinning) activity when administered to rats in a single dose [51], and it could depress aniline hydroxylase activity. Ethylmorphine demethylase, hepatic DNA, reduced glutathione and glucose-6-phosphatase were unaffected by chalepin (**1**) treatment at a dose of 50 mg/kg for 3 days prior to sacrifice. This coumarin also resulted in α-1-globulin increase and a decrease in β-globulin content of the serum. Intraperitoneal treatment with chalepin (100 mg/kg) for 2 days caused death of 4 rats out of 100 within 48 h of treatment. Livers of dead rats showed generalized necrosis of hepatocytes. Chalepin (**1**) induced alterations in the serum protein pattern within this period. Liver lesions were observed in chalepin treated animals and were characterized by mild necrosis of hepatocytes. However, no report on mutagenicity of chalepensin (**2**) is available to date.

#### **6. Drugability' of Chalepin (1) and Chalepensin (2)**

"Drugability" can simply be defined as the ability of a compound to be used as a pharmaceutical drug. In order for a molecule to be developed as a drug, it must have certain physicochemical characteristics, which can be measured or predicted by various experimental or mathematical models. The Lipinski rule of five, formulated in 1997 by Christopher A. Lipniski, can be used, albeit not conclusively, to predict whether a compound could be an ideal candidate as a drug molecule, i.e., whether a compound possesses "druglikeness" or not [57]. This rule states that an orally active drug does not have more than one violation of the following criteria: a molecular mass less than 500 Daltons, no more than five hydrogen

donors, no more than 10 hydrogen bonds and an octanol-water partition coefficient (log *P*) that does not exceed five. Sometimes an additional criterion, "molar refractivity should be between 40–130" is also added to the above rule. If we consider these criteria in relation to chalepin (**1**) and chalepensin (**2**), both compounds tend to follow Lipinski rule of five, and there is no violation of this rule whatsoever (Table 3), which suggests that these compounds possess "druglikeness" or "drugability" and have the potential for further development as commercial drugs. However, it must be noted that this rule of five was originally presented to aid the development of orally bioavailable drugs and was not intended for guiding the medicinal chemistry in the development of all small-molecule drugs. Moreover, there is hardly any reliable experimental bioavailability data available on these coumarins (**1** and **2**) to make any connections between bioavailability and the predicted values for the criteria shown in Table 3.

**Table 3. "**Druglikeness" of chalepin (**1**) and chalepensin (**2**) \*.

\* Data obtained from ChemSpider (www.chemspider.com, (accessed on 24 February 2021)) and DrugBank (https://go.drugbank.com/drugs/DB02205, (accessed on 24 February 2021)).

#### **7. Conclusions**

The present work generated the first comprehensive and critical review of published literature on chalepin (**1**) and chalepensin (**2**), revealing various bioactivities of these compounds and their potential as new therapeutic agents. Among the activities, it appeared that antiprotozoal, antiviral and particularly anticancer activities bear promises for these compounds for further consideration for development as therapeutic agents, when considered in the light of nonviolation of the Lipinski rule of five and low level of toxicities. However, there is no report on any systematic preclinical or clinical trial with these compounds involving human volunteers available in the literature to date. Therefore, further studies, including controlled preclinical and clinical trials, are still needed before we can comment on the true therapeutic potential of these compounds.

**Author Contributions:** All authors contributed equally to collation of relevant information from extensive literature search. Additionally, L.N. and S.D.S. prepared, edited and submitted the manuscript as corresponding authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** Lutfun Nahar (L.N.) gratefully acknowledges the financial support of the European Regional Development Fund—Project ENOCH (No. CZ.02.1.01/0.0/0.0/16\_019/0000868).

**Data Availability Statement:** All relevant data have been presented as an integral part of this man.

**Acknowledgments:** Lutfun Nahar gratefully acknowledges the financial support of the European.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Review* **Coumarin Derivatives in Inflammatory Bowel Disease**

**Luiz C. Di Stasi**

Laboratory of Phytomedicines, Pharmacology, and Biotechnology (PhytoPharmaTech), Department of Biophysics and Pharmacology, Institute of Biosciences, São Paulo State University (UNESP), 18618-689 Botucatu, SP, Brazil<EMAIL_ADDRESS>
**Abstract:** Inflammatory bowel disease (IBD) is a non-communicable disease characterized by a chronic inflammatory process of the gut and categorized into Crohn's disease and ulcerative colitis, both currently without definitive pharmacological treatment and cure. The unclear etiology of IBD is a limiting factor for the development of new drugs and explains the high frequency of refractory patients to current drugs, which are also related to various adverse effects, mainly after long-term use. Dissatisfaction with current therapies has promoted an increased interest in new pharmacological approaches using natural products. Coumarins comprise a large class of natural phenolic compounds found in fungi, bacteria, and plants. Coumarin and its derivatives have been reported as antioxidant and anti-inflammatory compounds, potentially useful as complementary therapy of the IBD. These compounds produce protective effects in intestinal inflammation through different mechanisms and signaling pathways, mainly modulating immune and inflammatory responses, and protecting against oxidative stress, a central factor for IBD development. In this review, we described the main coumarin derivatives reported as intestinal anti-inflammatory products and its available pharmacodynamic data that support the protective effects of these products in the acute and subchronic phase of intestinal inflammation.

**Keywords:** inflammatory bowel disease; coumarin; isocoumarin; Crohn's disease; ulcerative colitis; glutathione; oxidative stress; complementary therapies; intestinal inflammation

#### **1. Introduction**

Currently, the search and discovery of new drugs with efficacy, safety, and quality control to prevent and treat non-communicable diseases is a huge challenge for the chemical and pharmaceutical sciences as well as medicine. This task is not only a challenge in the present time but also to guarantee health quality for the next generations. Noncommunicable diseases, also known as chronic diseases, are persistent illnesses generally without a pharmacological cure, that tend to be of long duration, requiring long-term and systematic treatment approaches, and the result from multifactorial etiological factors. In general, patients with non-communicable diseases live throughout their lives with several symptoms, continuously using drugs to relieve them. Non-communicable diseases are a group of chronic disorders including cardiovascular diseases, diabetes, multiple sclerosis, obesity, arthritis, asthma, Parkinson's and Alzheimer's diseases, cancer, and inflammatory bowel disease (IBD), which have a high impact on the health system. According to a recent study, 56 million people died in 2017 and non-communicable diseases account for more than 73.4% of these global deaths, i.e., 41.1 million people [1]. Based on this, the search for new drug development to relieve symptoms and mainly to prevent non-communicable diseases is an important approach to improve several world health problems and patients' life quality.

The discovery of new drugs is based on synthetic chemistry, partial synthesis or modification of active molecules of synthetic or natural origin, and bioprospection of natural products, particularly from fungi and plant species. The research with natural products was overshadowed by the advent of the new technologies, synthesis of several

**Citation:** Di Stasi, L.C. Coumarin Derivatives in Inflammatory Bowel Disease. *Molecules* **2021**, *26*, 422. https://doi.org/10.3390/ molecules26020422

Academic Editor: Maria João Matos Received: 28 November 2020 Accepted: 23 December 2020 Published: 15 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

chemical active compounds, and international regulatory systems for biodiversity access established by the United Nations Convention on Biological Diversity. However, all current difficulties did not reduce the importance of the world biological biodiversity, particularly from tropical areas, as an inexhaustible and magnificent source of new medicines, which should be carefully and legally studied, respecting international regulatory systems and the traditional knowledge from local communities.

Natural products from plant and fungi origin are the source of several drugs with wide applications and pharmacological importance. Some of these compounds have defined the way of science and modern medicine as well as represented the basis of the treatment of several serious diseases and health problems affecting the world's people. The antibiotic penicillin discovered in *Penicillium* genus fungi; morphine, an opioid compound useful as a pain reliever, isolated of the opium plant (*Papaver somniferum*), and acetylsalicylic acid, a lead compound of the non-steroidal anti-inflammatory drugs, which is related to salicin, obtained from plants belonging genera *Salix* and *Populus*, are some emblematic examples of the natural products that have changed the history of medicine. Even with advanced modern medicine and biotechnology, the most recent discoveries of lead compounds include two products of plant origin, artemisinin and taxol, an antimalarial and an antineoplastic agent, respectively. The research related to the discovery of artemisinin from *Artemisia annua* received the 2015 Nobel Prize in Physiology or Medicine. Artemisinin completely changes the control of malaria and represents a new class of antimalarial drugs, whereas taxol, isolated from species belonging to the genus *Taxus*, particularly *Taxus brevifolia*, represents a new class of anti-cancer drugs. The number of lead compounds obtained from nature is high, showing that natural products play a key role in human health surveillance and represent the support basis of drug research and discovery.

Plant-based products are rich in several chemical classes of compounds, among which the alkaloids, terpenoids, tannins, and phenol and polyphenol compounds stand out, which are potentially useful to prevent and treat several disorders, particularly non-communicable diseases. Phenol and polyphenolic compounds, one of the most important classes of secondary metabolites from plants, include a plethora of different classes of molecules with high pharmacological value, among which the flavonols, flavanones, flavones, anthocyanidins, xanthones, stilbenes, catechins, quinones, and coumarins may be highlighted. These compounds represent an important source of new molecules with several pharmacological properties and are widespread in vegetables commonly consumed daily as dietary foods and spices. Dietary intake of several plants containing these compounds contributes to the plasma bioavailability of active molecules, which are useful both to improve immune response and act as preventative products for several non-communicable diseases. Nowadays, it has been considered that a properly used nutritional approach might be a part of the treatment of non-communicable diseases, particularly patients with Crohn's disease and ulcerative colitis, two chronic inflammatory disorders of the gut [2]. The pharmacological properties of phenol and polyphenol compounds against the inflammatory processes of the gut have been exhaustively reported, focusing on flavonoids [3,4], proanthocyanidins and anthocyanins [5,6], and catechins [7]. However, there is a lack of data and analysis of the potential use and application of coumarin and their derivatives as preventative and curative compounds in non-communicable diseases, particularly in inflammatory bowel diseases (IBDs).

In this review, we aim to update and systematize the available knowledge on the pharmacological activities of coumarin derivatives in the various in vivo experimental models of intestinal inflammation and in vitro studies to provide data and insights to further preclinical, clinical, and molecular studies, demonstrating the main and potential active coumarins useful to prevent or treat inflammatory bowel diseases as well as its main mechanisms of action and signaling pathways.

#### **2. Coumarin and the Main Coumarin Derivatives**

Coumarins, also known as benzopyrones, comprise a class of cinnamic acid-derived phenolic compounds found in fungi, bacteria, and plant species, particularly in edible, medicinal, and spice plants from different botanical families [8]. Coumarins are secondary plant heterocyclic metabolites composed of fused benzene and α-pyrone rings (Figure 1), and they occur widely in different parts of plants, such as roots, seeds, nuts, flowers, and fruits either as heterosides or in free form [8,9]. The term coumarin originated from de name "cumaru" the local name for the Brazilian teak plant (*Dipteryx odorata* Wild.) from the Fabaceae botanical family, in the traditional medicine of the Brazilian Amazon forest. *Dipteryx odorata* is an endemic plant of Central America and the North of South America, widespread in the Amazon Forest region, from which the coumarin was firstly isolated by Vogel in 1820 [10]. Its seeds, named tonka beans, are the natural source of coumarin, a compound widely used by the perfumery companies to replace vanilla, particularly as a fixative and enhancing agent in perfumes as well as added to toilet soap, detergents, toothpaste, tobacco, and alcoholic products [9]. Moreover, the isocoumarin, also recognized as 1*H*-benzopyran, is an isomer of the basic structure of coumarin, in which the orientation of the lactone ring is reversed (Figure 1). From the different substituted groups on the basic structure of isocoumarin, several subclasses and isocoumarin derivatives are also found in plant species such as paepalantine, capillarin, and thumberginol A (Figure 1). α‐ ‐

‐

**Figure 1.** Basic structures of coumarins and isocoumarins and the main isocoumarin derivatives. Chemical structures were drawn using ACD/ChemSketch software.

Coumarins are categorized into four main subtype classes of compounds: simple coumarins, furanocoumarins, pyranocoumarins, and the pyrone-substituted coumarins. Simple coumarins are composed of molecules with hydroxyl, alkoxyl, and alkyl substitution patterns on the basic structure and their glucosides [11]. Simple coumarin class represents the main class of coumarin derivatives with intestinal anti-inflammatory properties, particularly esculetin, esculin, 4-hydroxycoumarin, osthole, and 4-methylesculetin (Figure 2). ‐ ‐ ‐ ‐

Furanocoumarins are composed of coumarins derivatives in which a furan ring is fused with the basic structure of coumarin via C6-C7 or C7-C8 [10,11], generating linear furanocoumarins (fusion via C6-C7) such as psolaren, imperatorin, and xanthotoxin or angular furanocoumarins (fusion via C7-C8) such as isobergapten and angelicin (Figure 3). Similarly, in the pyranocoumarins, a six-membered pyran ring is fused with the benzene ring of the basic structure of coumarins via C6-C7 or C7-C8 [10,11]. Decursin (a linear pyranocoumarin) and seselin (an angular pyranocoumarin) are some examples of pyranocoumarins (Figure 3), which have no intestinal anti-inflammatory activity.  Chemical structures of the main simple coumarin derivatives with intestinal anti-inflammatory activity. Chemical structures were drawn using ACD/ChemSketch software.

Finally, pyrone-substituted coumarins are coumarin derivatives containing different chemical radicals fused with the pyran ring of coumarin [10,11]. Pyrone-substituted coumarins include natural and synthetic coumarins such as warfarin and dicoumarol (Figure 3). Pyrano-substituted coumarins have no intestinal anti-inflammatory activity but comprise some compounds with high pharmacological relevance. Warfarin is a derivative of dicoumarol, a pyrano-substituted coumarin isolated from hay species (*Melilotus alba* and *Melilotus officinalis*) after natural oxidation by several fungi, mainly *Penicillium nigricans* and *Penicillium jensi* found in moldy hay [12]. Dicoumarol and warfarin were first used as rodenticides due to their ability to promote internal hemorrhage in rodents [12]. The anticoagulant properties of dicoumarol and warfarin were the basis for the development of anticoagulant drugs to prevent stroke in patients with cardiovascular diseases, mainly atrial fibrillation and valvular heart disease, and to prevent and treat vein thrombosis and pulmonary embolism [13].

‐ **Figure 3.** Chemical structures of angular and linear furanocoumarins, angular and linear pyranocoumarins, and pyranosubstituted coumarins. Chemical structures were drawn using ACD/ChemSketch software.

#### ‐ **3. Inflammatory Bowel Diseases: General Aspects**

‐ ‐ ‐ ‐ Inflammatory bowel disease (IBD) consists of Crohn's disease (CD) and ulcerative colitis (UC), two relapsing chronic inflammatory processes of the gastrointestinal tract, which are part of a group of immune-mediated inflammatory diseases, without a definitive pharmacological treatment and cure [14]. Patients with CD or UC live with several harmful effects in their daily physical, social, and professional activities because these diseases produce limiting effects such as changes in intestinal habits with several evacuations, abdominal pain, diarrhea, bleeding, perianal fistulas and other extraintestinal manifestations.

‐ IBD is a disease that is increasing globally, affecting some 6 to 8 million people in the world and presenting a prevalence rate of 84.3 people (79.2 to 89.9) per 100,000 population in 2017 [15]. Although it is a disorder with low mortality and with a death rate of 0.51 per 100,000 population, IBD is growing exponentially around the world, showing the highest prevalence rates in North America and the United Kingdom and other European countries such as Norway, Poland, and Slovakia, whereas lower prevalence rate has been reported in several countries of Africa, South America, and Southeast Asia [15]. There is a direct relationship between the high prevalence rate of IBD and the industrialization level of a specific country, but the prevalence and incidence rates notably are also rising in newly industrialized countries [16]. The prevalence and incidence rate increment in developing or newly industrialized countries is associated with the industrialization process and migration of population from rural to urban areas, which promote changes in the lifestyle and the people choices related to diet, daily activities, and social behaviors. These changes suggest rates of IBD prevalence and incidence should thrive in parallel to those in the industrialized countries [16].

The IBD etiology is unclear, but several triggering factors have been related to its occurrence and development, including dysregulated immune response, dysfunctional intestinal barrier function, genetic, and environmental aspects. Currently, the pathogenesis of IBD involves a dysregulated autoimmune response and increased intestinal permeability related to gut dysbiosis, which is accelerated by exposure to environmental factors in individuals who have a pre-existing high-risk genotype [17]. The complex and inexact etiology of IBD is a limiting factor in the discovery of new pharmacological and complementary

therapies, and the development of preventive strategies useful to define a general protocol of treatment and definitive management of IBD patients. The multifactorial aspects of IBD also explain the high frequency of patients who are refractory to current pharmacological treatments, including conventional drugs such as aminosalicylates, glucocorticoids, immunosuppressants, and biological therapies based on monoclonal antibodies [18]. Current pharmacological treatment of IBD is based on the relief or to create a time of deep remission of symptoms. However, the long-term use of these drugs, produces serious side effects, reducing patient adherence to pharmacological treatment.

IBD also includes a lot of risk factors with an imbricated relationship among them. A series of interactions among risk factors, which do not act in isolation because none of the risk factors alone is sufficient for IBD development, have been suggested [19]. Risk factors for IBD development include intrinsic and extrinsic factors. The intrinsic factors involve genetic predisposition and familiar history as well as the intestinal microbiota, whereas extrinsic risk factors include smoking, appendectomy, hygiene, infections, use of antibiotics and other drugs such as NSAIDs (Non-steroidal anti-inflammatory drugs) and oral contraceptives, a diet with lower fiber and higher fat, vitamin D deficiency as well as lifestyle and social behavior, mainly high stress, sleep privation and lower physical activity [19]. Genetic and epigenetic studies have been extensively used as an important source of data, which are important for a better prediction of IBD course, identification of loci and candidate genes yielding valuable insights into the pathogenesis of IBD and disease pathways, which can be relevant in the clinical practice [20,21].

Intestinal microbiota, which has a key role in the pathogenesis of IBD, is an intrinsic factor that can be modulated by a series of products able to differentially affect distinct microorganisms, including functional food products, mainly probiotic, prebiotic, and symbiotic, natural products such as polyphenol compounds and standardized phytomedicines. In health conditions, intestinal microbiota via fermentation of dietary components produces a series of metabolites, mainly short-chain fatty acids (SCFAs), which are a source of energy for colonocytes and bacteria, and play several protective effects in the body after prompt absorption (Figure 4). On the other hand, the management of extrinsic factors, including changes in lifestyle, social behavior, and diet options as well as a lower exposition to other extrinsic factors can represent an important approach to reduce IBD development.

The combinatory action among genetic predisposition, external environmental factors, and intestinal microbiota is essential to the development of the dysregulated immune response and dysfunctional intestinal barrier [22], which are responsible for IBD development (Figure 4). Both CD and UC patients exhibit a dysfunctional intestinal epithelial barrier with increased permeability as well as an exaggerated immune response in the gastrointestinal tract towards the intestinal microbiota, which is not appropriately controlled, leading to intestinal inflammation [3,22]. The increase of intestinal permeability has been recognized as an early feature of the intestinal inflammatory process, which reduces the intestinal barrier function, a key factor to maintain intestinal homeostasis [23]. In this process, several factors and mediators are involved, particularly the zonulin pathway activation, a key process to control intestinal permeability, suggesting zonulin as a biomarker of gut permeability as well as a key target for the action of intestinal anti-inflammatory products [23–25]. The dysregulated immune response includes an innate immune response, the first-line defense against any damage promoted by pathogens, which is mediated by different cell types including macrophages, neutrophils, monocytes, dendritic, epithelial, and endothelial cells [3,22]. These cell types are responsible for phagocytosis, elimination of pathogens, production of several cytokines, and development of barrier and transport functions (Figure 4). The response to pathogens includes prompt participation of the antigen-presenting cells (APCs), which mediate the differentiation of T-cells into effector T helper (Th) cells, including Th1, Th2, and Th17 cell types, and regulator T-cells (Treg) (Figure 4), which are constituents of the adaptative immune response [3,22]. These different cell types are responsible for the synthesis and dysregulated release of a series of immunologic and inflammatory mediators with wide importance in the pathogenesis of

IBD, including a plethora of chemokines and cytokines. The pathophysiology of intestinal inflammation is very complex, including a wide number of signaling pathways and endogenous mediators as illustrated in Figure 4, where are particularly indicated the main targets for the action of coumarin derivatives.

‐

‐

‐

‐

‐

**Figure 4.** The main pathways of the intestinal inflammatory process or the action of coumarin derivatives. GS ‐ **Figure 4.** The main pathways of the intestinal inflammatory process or the action of coumarin derivatives. GSH, glutathione; IL, interleukin; iNOS, inducible nitric oxide synthase; MPO, myeloperoxidase; SCFAs, short-chain fatty acids; TGF, transforming growth factor; Th, T helper cells; Treg, T regulatory cells; TNF, tumor necrosis factor.

> Moreover, in the gastrointestinal tract, there is homeostasis of intestinal microbiota with immune cells responsible for a balance of host defense and immune tolerance, which can be shift leading to a dysbiosis process that plays a key role in the pathogenesis of IBD [17]. Although dysregulated immune response associated with both genetic predisposition and intestinal microbiota participates in the intestinal inflammatory process, oxidative stress characterized by an excessive release of reactive oxygen and nitrogen species (ROS/RNS) plays a key role in IBD pathogenesis [26,27]. The excess of oxidative mediators reacts with cell membrane fatty acids, proteins, and DNA impairing their functions (Figure 4). The production of superoxide, the main source of free radicals, is required to kill bacteria, a process that occurs especially in neutrophils and other cell types such as epithelial cells from the intestine. From superoxide production, several free radicals are produced via nitric oxide synthase and glutathione-related enzymes (Figure 4).

#### **4. Intestinal Anti-Inflammatory Activity of Coumarin Derivatives**

The dysregulated immune and oxidative response triggered by intestinal inflammation is an imbricated and complex interaction involving a series of endogenous mediators from different signaling pathways and receptors, such as nuclear factor-kappa b (NF-κB), nuclear factor erythroid 2 (NEF2)-related factor 2 (Nrf2), peroxisome proliferator-activated

receptor gamma (PPAR-γ), pregnane X receptor (PXR), hypoxia-inducible factor (HIF), several enzymes, especially cyclooxygenase 2 (COX-2), mitogen-activated protein kinases (MAPKs), and HIF-prolyl hydroxylases (PHDs) as well as mediators of intestinal epithelial barrier function such as zona occludens 1 (ZO-1), occludin, mucins (MUC1, MUC2, MUC 3, MUC4), and E-cadherin. These different pathways will be discussed in this review to explain the effects of some coumarin derivatives and their partially elucidated mechanisms of action. However, until now the majority of coumarin derivatives produce intestinal anti-inflammatory activity acting as modulators of oxidative stress and immune response, whereas a little number of coumarin derivatives were reported to act by other signaling pathways, which are also partially involved in oxidative stress modulation.

The intestinal anti-inflammatory activity of different coumarin derivatives was described using different experimental models of intestinal inflammation, mainly trinitrobenzene sulphonic acid (TNBS) and dextran sodium sulfate (DSS) in rats or mice as well as several in vitro studies with distinct cell types. Although coumarin is a class of natural and synthetic compounds with high chemical diversity, the intestinal anti-inflammatory has been limited to three subclasses of coumarin derivative, i.e., isocoumarins having one active compound namely paepalantine, a lot of simple coumarin derivatives, and the furanocoumarin imperatorin.

#### *4.1. Effects of Coumarin Derivatives on Oxidative Stress*

A recent review reported that different antioxidant compounds act by six general mechanisms: inhibiting free radical production by activated oxygen metabolites, changing the structural organization of free radical, producing a local decrease of oxygen concentration, interacting with organic radicals, chelating metal ions, and converting peroxides to stable and inactive products [28]. These processes reduce the availability of free radical species, improving the response against oxidative stress, which are the main properties of the coumarin derivatives with intestinal anti-inflammatory activity.

Oxidative stress is considered an imbalance in which excessive levels of oxygen free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) are present in the biological system in the face of inadequate availability of the antioxidants, which are capable to destroy these harmful products from metabolic processes [29]. A free radical is a molecule of the normal metabolism of oxygen with an unpaired or odd number of electrons, which is highly reactive and able to react with lipids from the cell membranes, proteins, and nucleic acids, affecting their structure and functions. In an inflammation process, ROS and RNS production is a prompt defense response of the body to kill several invading pathogens as well as to regulate the immune response via pro-inflammatory chemotaxis induction into the site of inflammatory processes as well as modulate the interactions and activation of the immune cell types [30]. However, when occurs an imbalance between free radical production and endogenous antioxidant response, the reaction of reactive oxygen and nitrogen species with host lipids, proteins, and nucleic acids generate oxidative stress, triggering a series of molecular and cellular events such as tissue damage and fibrosis [30], which are related to the origin and development of several chronic diseases, including IBD.

The reactive oxygen species and the endogenous mediators of antioxidative response are represented in Figure 5. The main reactive species in the biological system responsible for oxidative stress is the superoxide radical anion (O2 •−), which is formed by the addition of a single electron in molecular oxygen (O2) by the action of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) enzyme [26,27]. O2 •− is the source of hydrogen peroxide (H2O2), formed by the action of superoxide dismutase (SOD) enzyme, which catalyzes the dismutation of O2 •− into H2O2. Although H2O2 is not a free radical, it is a molecule highly reactive [29,30] and represents a key substrate for free radical production. H2O2 via Fenton reaction generates hydroxyl radical (OH−), which is the most reactive and harmful metabolite of oxygen metabolism. Simultaneously, H2O2 by the action

of myeloperoxidase (MPO) produces hypochlorous acid (HOCl), a potent antioxidant with antimicrobial activity and useful as a defense against several infectious pathogens [30,31].

MPO is the most toxic enzyme found in the granules of neutrophils and monocytes, two important cell types that participate in the intestinal inflammatory process with a key role in the innate immune response to pathogens [30]. MPO generates reactive intermediates, inducing oxidative peroxidation of lipids, and proteins and DNA damage. During the inflammatory response, MPO is released from neutrophils and monocytes, catalyzing the formation of HOCl from H2O2 (Figure 5). HOCl produced by the action of MPO influences the conversion of glutathione (GSH) to oxidized glutathione (GSSG), which disrupts the cellular redox balance, reducing the antioxidant GSH pool (Figure 5) and increasing the susceptibility to oxidative stress [30]. Generally, MPO activity is strongly increased in experimental models of intestinal inflammation, whereas simultaneously is possible to observe a depletion of GSH [32]. − − − −

‐ γ‐ γ‐ ‐ ‐ ‐ **Figure 5.** Free radical production and glutathione (GSH) antioxidant system. CAT, catalase; GPX, glutathione peroxidase, GR, glutathione reductase; GS, glutathione synthetase; GST, glutathione S-transferase; NOX, NADPH oxidase; MPO, myeloperoxidase; SOD, superoxide dismutase; γ-GLC, γ-glutamylcysteine ligase. In these oxidative processes, the following activities were reported: A. Counteraction of GSH depletion by paepalantine, coumarin, 4-hydroxycoumarin, esculetin, 4-methylesculetin, daphnetin, esculin, scopoletin, scoparone, and fraxetin; B. Inhibition of MPO activity by paepalantine, esculetin, 4-methylesculetin, daphnetin, and esculin; C. Scavenging activity of free radical by paepalantine, daphnetin, esculin, scopoletin, scoparone, and fraxetin; D. Inhibition of lipid peroxidation by esculetin, and daphnetin; E. Inhibition of GPX activity and expression by 4-methylesculetin; F. Increase of GST and GR activity and expression by 4methylesculetin. Chemical structures were drawn using ACD/ChemSketch software.

GSH, a tripeptide formed by glutamic acid, cysteine, and glycine, is the key endogenous antioxidant that participates in antioxidant response. GSH is produced by a reaction with two steps (Figure 5). Firstly, a residue of glutamic acid (Glu) binding with cysteine via γ-glutamylcysteine ligase (γ-GCL) action, producing γ-glutamylcysteine [33]. Secondly, γ-glutamylcysteine reacts with a residue of glycine under the action of glutathione synthetase (GS) enzyme to produce GSH [33]. GSH participates in antioxidant response acting as a free radical scavenger, reducing dehydroascorbate to ascorbate, which regenerates α-tocopherol from the α-tocopherol radical oxidation, and serving as a co-substrate for several antioxidant enzymes, mainly glutathione S-transferase (GST) and glutathione peroxidase (GPx) [34]. High pools of GSH is essential to modulate oxidative stress, and GSH also can be regenerated by oxidation of its disulfide-oxidized dimer (GSSG) by the action of glutathione reductase (GR) (Figure 5). This reaction occurs at expense of the reduction of NADPH from the pentose phosphate pathway [34].

The modulation of oxidative stress for the body's defense against free radical tissue damage and macromolecules oxidation is modulated by antioxidants, which are classified into nonenzymatic antioxidants consisting of micronutrient components, and enzymatic endogenous system [17,29]. The nonenzymatic antioxidant system includes several small molecules, mainly GSH, vitamin E, vitamin C, β-carotene, retinol, uric acid, and ubiquinol as well as several microelements like selenium, iron, zinc, copper, and manganese [29]. Vitamins act as donors and acceptors of ROS and the micronutrients act as cofactors, which regulate the activities of the antioxidant enzymes [29]. Endogenous enzymatic antioxidants involve superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST), glutathione reductase (GR), and γ-glutamyl transferase (γ-GT) enzymes [32], which act by different pathways to reduce the free radical availability and control oxidative stress.

In this process, CAT can break down two H2O2 molecules generating one molecular oxygen and two molecules of water [35], reducing H2O2 pool availability, which is used as a substrate for the production of OH− (Figure 5). In association with SOD and CAT, GPX, a dependent enzyme of micronutrient selenium, plays an important role in the reductions of H2O2 and lipid peroxides (LOOH) to produce water and lipid alcohol (LOH), contributing to modulation of oxidative stress and avoiding direct tissue damage [36]. Moreover, GST can catalyze the transfer of a GSH group to organic and inorganic electrophiles, reducing these compounds into unreactive products [34].

In another reaction pathway (Figure 6), nitric oxide synthase (NOS) controls the reaction of O2 •− with nitric oxide radical (NO• ) to produce free radical peroxynitrite (ONOO−), which is responsible for nitrosylation of proteins and oxidation of lipoproteins [31]. NO• is produced via enzymatic oxidation of L-arginine to L-citrulline by the action of constitutive and inducible NOS. Besides the mediator in blood pressure, NO• participates in the immune and inflammatory responses with biocidal activity against several microorganisms and induces damages on the proteins and DNA [31,37]. Toxic and oxidative effects of NO• results from its oxidation, generating highly reactive species, such as nitrite (NO2 −) and peroxynitrite (ONOO−).

NO2 − is produced by NO• autooxidation forming nitrous anhydride (N2O3), an intermediate in this conversion recognized as a potent nitrosating agent [31], which can also be used to produce nitrogen dioxide by the action of the MPO enzyme (Figure 6). Carbon dioxide (CO2) reacts catalytically with ONOO− to produce nitroperoxycarbonate (ONOOCO2), which via homolysis of the O-O bonds, carbon trioxide (CO3 •−), and nitrite dioxide (NO2 • ) radicals are produced [31]. Moreover, when ONOO- decomposes in the absence of CO2, the NO2 • and OH• radicals production take place, whereas, in the presence of CO2, CO3 •− and NO2 • radicals are produced, and in this process (Figure 6). MPO also participate, affecting tyrosine nitration when NO2 − is used as a co-substrate, with consequent production of NO2 • , a reactive free radical [31]. Based on its diverse action as a marker of neutrophil infiltration, inflammatory process, and oxidative stress, MPO represents a potential target for the development of synthetic and natural compounds against several diseases, including atherosclerosis, acute coronary syndromes, ischemic heart disease, and IBD [30,38,39].

− −

**Figure 6.** Nitric oxide synthase (NOS) pathway of free radical production. NOX, NADPH oxidase; NOS, nitric oxide synthase; MPO, myeloperoxidase. In these oxidative processes, the following activities were reported: A. Inhibition of iNOS activity by paepalantine, esculetin, esculin, auraptene, and collinin; B. Reduction of NO release by isomeranzin and esculin.

> − − ‐ − ‐ − − ‐ The major regulator of the endogenous antioxidant system is the nuclear factor erythroid 2 (NEF2)-related factor 2 (Nrf2) that protects cells from several stressors agents, such as ROS, RNS, and environmental damage [40]. In physiological conditions, Nrf2 binds with cullin 3 (cul3) and Kelch-like ECH-associated protein 1 (keap1), a key repressor of the Nrf2 signaling pathway (Figure 7), preventing the translocation of Nrf2 to the nucleus [41]. This complex, after ubiquitination, promotes Nrf2 degradation via proteolysis. Under oxidative stress conditions, Nrf2-keap1 complex is uncoupled and a free Nrf2 is translocated into the nucleus, where binds with small Maf (sMaf) proteins [42]. The heterodimer binds with antioxidant response elements (ARE) target genes (Figure 7), regulating the expression of several antioxidant-related endogenous genes, including the enzymes CAT, GPX, SOD, GST, γ-GCL, GR, NADPH quinone oxidoreductase, and heme oxygenase [41]. Based on this, Nrf2 is a key mediator of the antioxidant defense system as well as an important target for the action of new synthetic and natural compounds, including coumarin derivatives such as esculetin.

> > ‐ ‐

‐

‐

‐ ‐ ‐ ‐ ‐ ‐ ‐ γ γ‐ ‐ **Figure 7.** The nuclear factor erythroid 2 (NEF2)-related factor 2 (Nrf2) signaling pathway of oxidative stress. ARE, antioxidant element of response; CAT, catalase; cul3, cullin 3; GST, glutathione S-transferase; GPX, glutathione peroxidase; GR, glutathione reductase; HO-1, heme-oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2; sMaf, small Maf proteins; Ub, ubiquitin; γGCL, γ-glutamylcysteine ligase. In these oxidative processes, the following activities were reported: A. Upregulation of Nrf2 by esculetin, 4-methylesculetin, daphnetin, and esculin.

#### ‐ ‐ 4.1.1. The Isocoumarin Paepalantine

‐ ‐ ‐ ‐ ‐ ‐ ‐ μ Paepalantine (9,10-dihydroxy-5,7-dimethoxy-1*H*-naptho(2,3c)pyran-1-one), was the first plant-derived coumarin studied in an experimental model of intestinal inflammation [43]. Paepalantine (Figure 1) is an isocoumarin previously isolated from the capitula of the Brazilian endemic *Paepalanthus bromelioides* plant from the Eriocalulaceae botanical family [44], which produced protective effects in the acute and relapse phases of the intestinal inflammation induced by TNBS in rats [43]. The protective effects observed after oral administration of the 5 and 10 mg/kg were similar to those promoted by the 25 mg/kg of sulphasalazine, a 5-aminosalicylate currently used to treat human IBD, i.e., paepalantine produced intestinal anti-inflammatory activity at doses 2.5 and 5.0-times lower than a reference drug [43]. Intestinal anti-inflammatory activity of the paepalantine was related to prevention of the GSH depletion (Figure 5) and inhibition of the colonic NOS activity (Figure 6), which was upregulated by the inflammatory process, suggesting that intestinal anti-inflammatory activity is related to its antioxidant properties [43]. Paepalantine also inhibited HOCl production in rat neutrophils, reducing oxidative stress

by the inhibition of MPO activity and scavenging HOCl (Figure 5) [45]. Moreover, the antioxidant properties of paepalantine were evidenced by its potent scavenging properties in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide radicals as well as by its ability to protect mitochondria from hydroperoxide accumulation and mitochondrial membrane lipid peroxidation [46,47]. The inhibition of iNOS activity by paepalantine was recently corroborated through in vitro studies with LPS-stimulated macrophages [48]. In this study, paepalantine binds with the NOS enzyme through several structural amino acids just on the active site of the enzyme, reducing its enzymatical activity [48].

#### 4.1.2. Coumarin and 4-hydroxycoumarin

Intestinal anti-inflammatory activity of coumarin and its derivative 4-hydroxycoumarin (Figure 2) were evaluated in the acute and subchronic phases of the TNBS-induced intestinal inflammation model in rats [49]. In this study, damage score and extension of tissue lesion induced by TNBS were significantly reduced after oral administration of coumarin (25 mg/kg) and 4-hydroxycoumarin (10 and 25 mg/kg), and these protective effects were accompanied by a counteraction of GSH depletion and inhibitory MPO activity (Figure 5) [49]. Although 4-hydroxycoumarin produced effects at lower doses when compared with coumarin, is not possible to suggest that the OH substitution in C4 was directly related to the improvement of its effects in the acute or sub-chronicle protocols of intestinal inflammation induced by TNBS in rats.

#### 4.1.3. Esculetin (6,7-dihydroxycoumarin) and 4-methyl Esculetin