Halimanes and cancer: ent-halimic acid as a starting material for the synthesis of antitumor drugs

The development of new anti-cancer agents is an urgent necessity nowadays, as it is one of the major causes of mortality worldwide. Many drugs currently used are derived from natural products. Halimanes are a class of bicyclic diterpenoids present in various plants and microorganisms. Many of them exhibit biological activities such as antitumor, antimicrobial, or anti-inflammatory. Among them, ent-halimic acid is an easily accessible compound, in large quantities, from the ethyl acetate extract of the plant Halimium viscosum, and it has been used as a starting material in a number of bioactive molecules. In this work, we review all the natural halimanes with antitumor and related activities until date as well as the synthesis of antitumor compounds using ent-halimic acid as a starting material.


Introduction
Natural products (NPs) constitute an abundant and diverse source of chemical structures, which are currently used in the search of bioactive molecules and drug discovery (Karageorgis et al., 2021).In this manner, the study of natural products and their derivatives remains one of the most important research areas in organic, biological, and medicinal chemistry (Kumar and Waldmann, 2009;Karageorgis et al., 2021).
Among the last decades, natural product-like drugs have a higher success rate in showing bioactivity (Newman and Cragg, 2012).In fact, approximately half of the drugs in clinical use come from living organisms (Paterson and Anderson, 2005), and in the last few years, 73% of antitumor drugs were either natural products, bioinspired compounds, or natural product derivatives (Wilson and Danishefsky, 2006;Newman and Cragg, 2007).
This elevated bioactivity of NPs can be explained by the purpose of their biosynthesis itself (Danishefsky, 2010).Living organisms synthesize compounds in order to use them in their own metabolic pathways.Thus, these compounds fit in different kinds of proteins and enzymes involved in metabolic engineering.Hence, these compounds possess well-defined tridimensional structures rich in functional groups and adequately oriented in space, which can be used in drug modeling with a higher success rate.
Bioactive compounds are often asymmetric, and their biological activity is closely related to one of the enantiomers.However, there is also the possibility that both enantiomers show different activity.For this reason, the effective synthesis of homochiral molecules that are enantiomerically pure remains one of the biggest challenges that modern organic chemistry must face.Thus, in order to reach enantiomerically pure compounds, different methodologies can be applied from the kinetic resolution of racemic mixtures to the enantioselective synthesis using chiral natural products as starting materials (chiral pool) as well as the asymmetric synthesis using chiral auxiliaries, reagents, or catalysts for that purpose.
The synthetic approach to bioactive compounds by the transformation of easily accessible and abundant natural products (chiral pool) is widespread.The use of these natural products in the enantioselective synthesis of compounds of similar carbon backbones usually represent an advantage compared with the total synthesis in terms of economy of the process and synthetic steps.Many examples can be collected from the literature where chiral pools are used, but the examples of the synthesis of paclitaxel (Taxol ® ) (Gennari et al., 1996;Kingston et al., 2002) and ecteinascidin 743 (ET-743, Yondelis ® ) (Cuevas et al., 2000) are paradigmatic of this strategy.
Ent-halimic acid 1 belongs to the bicyclic diterpene family of halimanes (Urones et al., 1987;Roncero et al., 2018).The use of 1 as a starting material allowed access to a variety of compounds with biological interest, such as antibiotic, antifeedant, antitumor, antifouling, and antiviral..In this work, the use of ent-halimic acid 1 as a starting material in the synthesis of antitumor compounds is reviewed (Figure 1).Compound 1 is easily accessible in large quantities from the ethyl acetate extract of Halimium viscosum (Cistaceae).The vegetal source is widespread in the Iberian Peninsula, mainly in Spain (De Pascual Teresa et al., 1985;De Pascual Teresa et al., 1986;Urones et al., 1987) and Portugal (Rodilla et al., 1998;Rodilla et al., 2001).
The functionalization appearing in ent-halimic acid, with an allylic hydroxylic group in the side-chain and a Δ 1 (10) double bond in the bicyclic system as well as a carboxylic acid at C-18, confer to excellent characteristics of (1) for its use as a starting material in the synthesis of antitumor compounds.
We have divided this work in two main aspects: first, we will summarize all the known natural halimanes, which exhibit antitumor as well as antitumorigenic-related bioactivities, and finally, we will review all the synthesis of bioactive compounds using ent-halimic acid as a starting material.

Natural bioactive halimanes
Natural halimane skeleton compounds showing antitumor activity as well as antitumorigenic-related bioactivities are reviewed herein (Figures 2-5).These antitumor compounds have been classified into four groups according to their structural frameworks (data shown in parentheses represent the IC 50 value of the corresponding compound).
• Cajucarinolide 48 and isocajucarinolide 49 are potent PLA 2 inhibitors with IC 50 of 5.8 and 2.3 μg/mL, respectively, (Ichihara et al., 1992).PLA 2 may be a target in cancer treatment because it is involved in pro-inflammatory and pro-tumoral pathways (Peng et al., 2021;Vecchi et al., 2021).• Crassifolins S-U (50-52) were isolated from Croton crassifolius and tested for anti-inflammatory and antiangiogenesis activities (Li et al., 2021).All of them were active, and crassifolin U 52 was the most active for both bioactivities.
3 Synthesis of antitumor terpenoids using ent-halimic acid as a starting material In this part of the work, the synthesis of a series of antitumor compounds using ent-halimic acid 1 as a starting material is described.

Synthesis of ent-halimanolides
Starting from ent-halimic acid 1, a series of natural enthalimanolides have been synthesized.These compounds are characterized by showing a lactone ring in any position of the halimane skeleton.The synthetic routes and biological evaluations are described for each case.

Synthesis of butenolides and γhydroxybutenolides
The synthesis of 40, 61, and 63 (Hara et al., 1995) using enthalimic acid methyl ester 59 as a starting material is performed according to the route showed in Scheme 1 (Marcos et al., 2003b;Marcos et al., 2005b).
The transformation of 59 of the intermediate methyl ketone 60 in six steps (Scheme 1) consists of C18 reduction and degradation of the carbon side chain.To access the key intermediate 40, the butenolide framework is attached in three steps by a reaction with Bestmann ketene to the corresponding αhydroxyketone of 60, yielding 40.For accessing 61, the doublebond isomerization is required, and in the case of 63, oxidation of C16 and double-bond isomerization is required (Boukouvalas and Lachance, 1998).

Furo-ent-halimanolide synthesis
Using 59 as a starting material (Marcos et al., 2008), furo-enthalimanolide 12 was synthesized (Scheme 2).The synthesis of 12 from ent-halimic acid methyl ester 59 uses aldehyde 65 as an advanced intermediate.The transformation of 59 into 64 consists of the degradation of the side chain and C-2 functionalization, and then the tetranoraldehyde 65 is synthesized by side chain shortening and C-2 functionalization, which in parallel causes the double-bond isomerization.The furyl fragment is coupled to the aldehyde 65 in the side chain.After that, lactonization and C-12 oxidation lead to 12 in good yield.
The biological assays carried out showed antitumor activity for compound 12 against HeLa cell lines ( Marcos et al., 2003b).

Sesterterpenolide synthesis
The study of marine metabolites has aroused great interest in recent years.Among these natural products, a considerable number of sesterterpenoids have been isolated (Faulkner, 2001;Blunt et al., 2003).Many of them possess a γ-hydroxybutenolide (Brohm and Waldmann, 1998;Demeke and Forsyth, 2000;Piers et al., 2000) moiety as a significant structural feature and, in many cases, are involved in their biological activities.
Cladocoran A and B (Figure 7) are sesterterpenolides, whose initially proposed structures were 71 and 72 (Fontana et al., 1998).The synthesis of these structures from ent-halimic acid, together with the spectroscopic data of the natural products named cladocoran A and B, led to the suggestion of a revision of their proposed structures as isoprenyl ent-halimanolides.

Synthesis of sesterterpenolides 71 and 72
Together with the synthesis of 71 and 72 (Marcos et al., 2002;Marcos et al., 2003a), the synthesis of the enantiomers and their C-18 epimers by Miyaoka et al. (2003) made it possible to establish the structure of the natural products cladocoran A and B as an olefinic analog of dysidiolide (cladocoran B) and its acetate (cladocoran A).
The synthesis of 71 and 72 using 59 as a starting material was accomplished according to the following retrosynthetic pathway (Scheme 3).
The synthesis of diol 75 (Scheme 4) is afforded by transformation in seven steps.First, the homologation and reduction of C18 position are performed, yielding intermediate 77, to which the side chain is degraded and adequately functionalized, leading to diol 75.The synthesis of aldehyde 74 from intermediate diol 75 is performed in four steps consisting of the shortening of the side chain in two carbons (C13 and C16) (Scheme 4).For that purpose, the following transformations are required: protection of the primary alcohol as an acetyl derivative and dehydration of the side chain alcohol; then, oxidation and cleavage of the resulting epoxide.
The reaction of aldehyde 74 with 3-furyllithium led to furo derivatives 81 and 82 (Scheme 5).Taking each C8 epimer separately, 81 and 82 are transformed in the corresponding isoprenyl-entfurohalimanes 83 (18S) and 73 (18R), respectively, by elongation of the carbon chain of the southern part in three steps.
Finally, the oxidation of furo derivatives 73 and 83 led to their corresponding γ-hydroxybutenolides 72 and 84.When C18 is first acetylated, the same procedure applied to compounds 71 and 85.The γ-hydroxybutenolides 71, 72, 84, and 85 can be decreased, respectively, to the corresponding butenolides 88, 86, 87, and 89.The interest in disposing of other synthetic analogs of dysidiolide for their antitumor activities led the obtention of these butenolidecontaining sesterterpenoids, readily available from their corresponding γ-hydroxyderivatives.
The cytostatic and cytotoxic properties in tumor cell lines (HeLa, HL-60, HT-29, and A549) of compounds 72, 84, 85, and 87 were determined showing IC50 in the range of 0.9-7.9μM.The results are comparable with dysidiolide (Takahashi et al., 2000) where the synthesized compounds improve the antitumor activity against some tumor cell lines.
Similar synthetic procedures were applied for the synthesis of new dysidiolide analogs (90-96), where the butenolide moiety appears in the south part and the isoprenyl unit is bonded as a carbon side chain (Figure 8).Other analogs (97) have been synthesized showing a tricyclic core system in which the γhydroxybutenolide is attached (Marcos et al., 2007).
The in vitro antitumor activity for these synthetic compounds was tested in HeLa, HL-60, HT-29, and A549 cell-lines.The capability of compounds 90-97 to inhibit tumor cell growth was significant in the low micromolar range.In this manner, these dysidiolide analogs are slightly more potent than proper dysidiolides (Marcos et al., 2007).

Synthesis of PUFAs and phospholipid hybrids with sesterterpenoids
Bioconjugate compounds have emerged in the last decades as a novel tool and therapeutic strategy in medicinal chemistry (Nilo et al., 2014;Lampkowski et al., 2015;Romero-Hernández et al., 2015;Shenvi et al., 2015;Zamudio-Vazquez et al., 2015).Bioconjugate molecules have been described as bioactive agents, showing a synergistic effect due to conjugation.
On the other hand, edelfosine is a well-known alkylether lipid that belongs to the so-called antitumor lipid family and is widely studied for its antitumor, antiparasitic, and other bioactivities (Mollinedo et al., 1997;Gajate and Mollinedo, 2002;Mollinedo et al., 2004).In addition, PUFAs as DHA and EPA show antitumor activity.In this way, synthesizing hybrid compounds of these antitumor molecules makes a promising approach for antitumor therapy.The following part describes the synthesis and biological results of the antitumor sesterterpenoid hybrids that are structurally related to dysidiolide with PUFAs and edelfosine analogs (Gunasekera et al., 1996;Corey and Roberts, 1997;Brohm et al., 2002a;Brohm et al., 2002b).
Taking the furyl and γ-hydroxybutenolide bioconjugates as examples for the synthesis of these kinds of compounds, the synthetic route can be divided in three steps: synthesis of the glycerolipid, conjugation with the sesterterpenoid fragment previously synthesized via carbonate linker, and finally, adequate functionalization of the glycerolipid either via phosphocholine polar head or EPA esterification.
A general synthetic scheme for the synthesis of the bioconjugates 104-111 is shown in Scheme 6 (Gil-Mesón et al., 2016).The preparation of the bioconjugate dealt here consists of three key steps: synthesis of the lipidic chain and its adequate functionalization for the hybridization step; synthesis of the sesterterpenic fragment, and finally, coupling of both fragments and derivatization for new analogs.
The glycerolipidic part of the molecule is readily prepared from R-solketal 100 in four steps to access the chlorocarbonate 99 that is adequate for the coupling with the sesterterpenoid 102/103 and yielding the hybrids showing the furan moiety.In these cases, the sesterterpenolide is bonded to the glycerol in sn2 (Gil-Mesón et al., 2016).
For preparing sn1-bonded sesterterpenoid from racemic glycerol, 100 is used as a starting material in the synthesis of the glycerolipid.The protected triol 100 is transformed into 101 in four steps, attaching a chlorocarbonate in sn1.The coupling to form the bioconjugate occurs in excellent yield.The esterification between EPA and a glycerol hydroxy group is performed to obtain the furobioconjugates (104, 105, and 110).The following two steps are required to attach a phosphocholine polar head to the glycerolipid: phosphorylation and choline esterification, leading to the corresponding glycerophospholipid bioconjugate (108).The transformation of the furylderivatives (104, 105, 108, and 110) into the corresponding hydroxybutenolides can be performed by oxidation with 1 O 2 and rose bengal, yielding 106, 107, 109, and 111, respectively.Different structural modifications have been introduced in the synthesis of the bioconjugated compounds, including synthesizing molecules showing the furyl or butenolide fragments in the north or south part, as well as bioconjugation with a PUFA directly via a carbonate group or bioconjugation with a PUFA through a glycerol unit attached to the carbonate linker (Figure 9).
The synthesized bioconjugates have been tested against different tumor cell lines (HeLa and MCF-7).The corresponding bioconjugates showed higher antitumor activity compared with their relative non-conjugate fragments, but simple bioconjugates 112 and 113 show higher activities compared with 108 and 109, respectively.The chirality of the glycerol unit does not seem relevant for the antitumor activity (Mollinedo et al., 1997;Samadder et al., 2004).When the γ-hydroxybutenolide moiety instead of the 3-furyl group is present in the sesterterpenoid scaffold, the antitumor activity is significantly enhanced, increasing even two orders of magnitude.For that reason, the γ-hydroxybutenolide fragments together with the bioconjugation are the highlighted structural frameworks of this family of molecules (106, 107, 109, 111, and 113).Although the attachment position of the sesterterpenoid  fragment in glycerol does not seem very important in terms of bioactivity, when the sesterterpenoid is attached at the sn2 position, the activity is slightly better compared to the sn1 substitution.

Synthesis of sesquiterpene quinone/ hydroquinone
The interest of these secondary metabolites (Figure 10) showing quinone/hydroquinone frameworks with drimane or rearranged drimane skeleton lies, mainly, in their biological activities and have been widely studied for this purpose (Capon, 1995;Marcos et al., 2010).
As antitumor agents, they show cytotoxicity in cancer cell lines such as HeLa, A549, HCT, KB16, P388, and Ehrlich cells, among others; moreover, antiviral, cytotoxic, hemolitic, anti-inflamatory, antiproliferative, antifeedant, antiplasmodial, antimalarial, cardiotonic antituberculosis, and antimicrobiane properties are also described.The biological assays studying the enzymatic inhibition have demonstrated the capability of different quinone/hydroquinone natural products to inhibit DNA topoisomerase I and II, tyrosine kinase, Ca 2+ /K + ATPase, Frontiers in Chemistry frontiersin.org10 PI3 kinase, and PTP1B as well as interleukin-8 dissociation of its receptors.
Using ent-halimic acid 1 as a starting material, different natural products (114)(115)(116) showing drimane or rearranged drimane skeleton have been synthesized (Talpir et al., 1994;Kurata et al., 1996;Fraga, 2003).The synthesis afforded for these compounds is shown in Scheme 7 and consists in the following key-steps: degradation of the side chain and C-18 reduction to obtain the tetranor intermediate 117; ring-D coupling and functionalization; and finally, cyclization if needed for ring C formation.
The transformation of ent-halimic acid 1 into tetranorhalimane 117 takes place in 11 steps with good overall yield (Scheme 7).Thiopyridine quinone 118 is prepared by the Barton decarboxylation and rearrangement.Quinone 118 is an intermediate for the synthesis of aureol 114 and the tri and tetracyclic quinones 115 and 116, respectively.For the synthesis of aureol 114 from 118, two steps are required: first, the reduction to hydroquinone 119 and then the cyclization in acidic medium, accessing aureol 114 in this way.On the other hand, the addition of two equivalents of sodium methoxide in two steps yield 120 that can be transformed into neomamanuthaquinone 115 by acidic hydrolysis with HClO 4 .Finally, menoqualone 116 is synthesized from 115 by reaction with p-TsOH, promoting its cyclization and ring-C formation.

Sesqui-and diterpene alkaloids
The use of ent-halimic acid 1 as a starting material for sesquiand diterpene alkaloids has been applied for the preparation of 7,9dialkylpurines (agelasine C) as well as di-and sesquiterpenyl indoles.
In particular, agelasine C (Figure 11) is one of the first four known agelasines isolated by Nakamura et al. (1984) from an Okinawan sea sponge of genus Agelas.The biological studies of (-)-agelasine C showed a high activity inhibiting Na + /K + ATPase and antimicrobial activities.
Later on, Hattori et al. (1997) isolated epi-agelasine C 112 from marine sponge Agelas mauritania as an antifouling active agent against macroalgae.
The comparison of the spectral data of compounds 121* and 122* with those of 123 suggested to correct the initially proposed structures to the revised structures (121 and 122) appearing in Figure 11.
Due to the interest on the biological properties of natural agelasine C and epi-agelasine C, the synthesis of 123 analog was carried out (Marcos et al., 2005a).The synthetic strategy used for accessing this meroterpenoid consists of the preparation of the diterpene fragment functionalized with bromine and then coupling with the purine derivative (Scheme 8).

Synthesis of sesquiterpenyl indoles
Terpenyl indoles are natural meroterpenes composed of indole alkaloids and terpenoids (Marcos et al., 2013a).Among these natural products, sesquiterpenyl indoles are a small group of natural occurring products of great interest due to their variety of biological properties, such as anticancer, antibacterial, or anti-HIV (Williams et al., 2010;Marcos et al., 2013a).The first sesquiterpenyl indole described was polyalthenol 128, isolated in 1976.After that, different natural products have been identified as sesquiterpenyl indoles, but pentacyclindole 129, isolated from G. suaveolens roots, was a new natural product showing a novel
Frontiers in Chemistry frontiersin.org13 Roncero et al. 10.3389/fchem.2023.1225355framework.Both natural product structures, 128 and 129, were corroborated as well as the absolute configuration determined by synthesis using ent-halimic acid 1 as a starting material, whose synthetic routes are described in the following section.

Synthesis of 12-epi-polyalthenol
In order to confirm the structure of polyalthenol 128, the synthesis of 130 and 131 was carried out (Marcos et al., 2012).
By the synthesis of both epimers at C19, that stereocenter was easily determined compared with polyalthenol.130 is the one that showed a similar NMR pattern for the signal corresponding to C19.The comparison of the spectroscopic data allowed to conclude that 130 is a C12 epimer of polyalthenol.That fact, together with the opposite rotatory power shown by these molecules led to define 130 as (-)-12-epi-ent-polyalthenol, confirming the structure of the natural product 128 as a

FIGURE 11
Initially proposed structures for agelasine C and epi-agelasine C as well as the revised structures corrected due to the synthesis of 123 from enthalimic acid, whose absolute configurations were known.
The biological studies of 130, 131, 135, and 136 were performed, where these compounds showed significant antitumor and antiproliferative activities against three cancer cell lines (A549, HL-60, and MCF-7).

Synthesis of 12-epi-ent-pentacyclindole 137
Pentacyclindole is a unique natural product due to its structure, being the only natural product known with its pentacyclic framework.The synthesis of its 12-epi diastereoisomer using enthalimic acid uses the previously synthesized indolederivative 130 as an intermediate (Scheme 10).The cyclization bonding C-2 and C-17 in this biomimetic synthetic route can be considered the key step in the preparation of pentacyclindole analogs.Pentacyclindoles 129 and 137 are epimers at C-12, and by synthesizing 137 from enthalimic acid 1, which is carried out with excellent yields, the absolute configuration of 129 was possible to be established (Marcos et al., 2013b).

Polyalthenol and pentacyclindole analogs as antitumor compounds
The synthesis and biological activity of polyalthenol and pentacyclindole analogs using similar synthetic routes, as shown for the synthesis of the epimer natural products, have been carried out (Marcos et al., 2014).A summary of all the compounds synthesized from ent-halimic acid 1 is displayed in Figure 12.
The structural modifications introduced in the analogs of polyalthenol can be divided into indole-ring functionalized, 2methylindoles, C-2 functionalized, and C-19 deoxygenated (Figure 12).
In the case of the synthesis of PLA 2 inhibitors, the most remarkable considerations are that the indole C-2 must be functionalized, and other requirements are the carboxylic acid as well as the nitrogen protected with a bulky group.
The antibacterial activity for these compounds is reported.The antiproliferative activities of these analogs have been tested in several tumor cell-lines (A549, HBL-100, HeLa, SW1573, T-47D, and WiDr).All compounds exerted inhibition of cell growth in the range of 1-70 μM.In terms of structural activity, it can be concluded from this study that the presence of substitutions in C-2 decreases the activity slightly (139-148 vs. 149-158).When substitution appears in the benzene ring of the indole either at C-6 or C-8, only the methoxy group enhances the activity (144 and 154).When a methyl group esterifies carboxylic acid in 161 resulting in a total loss of bioactivity, however, the free carboxylic acid in 162 recover the antitumor properties.
In the synthesis of pentacyclindole analogs, the appearing structural mosaics in the synthesized analogs of pentacyclindoles can be divided in the function of the position: indole-ring functionalized, 2,3-dihydrofuran, and C-19 deoxygenated derivatives.

Conclusion
In this article, we have put together a series of natural halimanes with antitumor activity evaluated against different pharmacological targets and in different cell lines.Structurally, the halimanes that exhibit antitumor activity can be classified into four groups: halimanes with acyclic side chain, 14,15furohalimanes, 14,15-halimanolides, and halimane-purine hybrids.
Moreover, we have reviewed the role of ent-halimic acid 1 as a starting material in numerous syntheses of bioactive compounds.This is a key aspect of this compound that confers it great importance, as it can be obtained easily from the extracts of Halimium viscosum, reducing time and effort for the obtention of more complex structures.Some of them, like the terpene-purine hybrids or the bioconjugates with antitumor lipids (edelfosine) or PUFAs, show promising results as cytotoxic and antitumor compounds, and more studies should be made to improve the bioactivities and understand the antitumor mechanism.
the manuscript, made corrections to the first draft, and wrote the final draft.All authors contributed to the article and approved the submitted version.

FIGURE 6
FIGURE 6Overview of the use of ent-halimic acid 1.