Indolactam Alkaloids: Syntheses and Emerging Therapeutic Applications

Abstract

Protein kinase C (PKC) enzymes play critical regulatory roles in maintaining cellular homeostasis. In turn, specific isoforms have emerged as targets for therapeutic intervention, and recent studies have highlighted the importance of PKC activation in preventing cancer cell proliferation. The indolactam alkaloids are a family of natural products that potently activate conventional and novel PKC isozymes. Importantly, this activity offers new opportunities for drug design and the development of novel chemotherapeutics. In this review, we describe our recent synthetic studies aimed at accessing numerous members of this natural product family. We also detail various approaches to prepare focused libraries of indolactam-based analogues. These efforts have facilitated medicinal chemistry studies of the indolactam scaffold and importantly provided a preclinical platform for evaluating PKC activation as a therapeutic strategy in multiple cancer models.

1 Introduction

2 Total Syntheses of Indolactam Alkaloids

3 Biological Studies of Indolactam Alkaloids

4 Conclusions and Outlook

 

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Key words

alkaloids - total synthesis - medicinal chemistry - therapeutics - protein kinase C

Biographical Sketches

Jordan MacQueen received his bachelor’s degree in chemistry from Loyola University Chicago. Currently, he is pursuing his doctoral degree at the Loyola University Chicago under the supervision of Prof. Kelvin L. Billingsley. His research interests include total synthesis and medicinal chemistry.

Kelvin L. Billingsley is an associate professor in the Department of Chemistry and Biochemistry at Loyola University Chicago. He obtained a doctoral degree in chemistry from the Massachusetts Institute of Technology and completed his postdoctoral training at Stanford University. His research interests are in the areas of medicinal chemistry, chemical biology, and hyperpolarized ¹³C magnetic resonance spectroscopy.

1

Introduction

Protein Kinase C (PKC) is a family of 10 Ser/Thr kinases that share a common catalytic domain but possess distinct regulatory domains.[1] These isozymes are divided into three classes: conventional (cPKCs: α, βI, βII, γ), novel (nPKCs: δ, ε, η, θ), and atypical (aPKCs: ι/λ, ζ). Both cPKC and nPKC isozymes bind the endogenous regulatory domain agonist diacylglycerol (DAG), whereas aPKC isozymes are DAG-insensitive.[2] The roles of PKCs in the cell are ubiquitous, including regulation of cell growth,[3] modulation of membrane structure,[4] and control of transcription;[5] in turn, these roles impact diverse physiological functions, ranging from learning and memory[6] [7] to immune responses.[8,9] Dysfunction of PKC isozymes is implicated in pathologies such as diabetes,[10,11] cardiovascular disease,[12] [13] Alzheimer’s disease,[14] and autoimmune disorders.[15] In addition, disruption of signaling pathways by dysregulated PKC activity is associated with aggressive tumor promotion in a variety of cancers.[16] [17] [18]

Numerous natural products (e.g., bryostatins and phorbol esters) have been discovered to act as highly potent DAG mimics, strongly activating cPKC and nPKC isozymes.[19] [20] [21] [22] [23] This bioactivity has provided novel opportunities for natural-product-inspired drug design as a strategy for therapeutic intervention in PKC-dependent disorders.[24–30] In addition, recent investigations have provided compelling evidence that PKC isozymes play a tumor-suppressor role in many cancers. This emerging feature of this enzyme family may offer new inroads for PKC agonists to serve as chemotherapeutics.[16,31]

The indolactam alkaloids are a class of over 40 natural products that generally possess high affinity for the DAG-binding site of cPKC and nPKC isozymes (Figure [1]).[32] (–)-Indolactam V (1) represents the core structure found in nearly all members of this family, as it possesses a tricyclic ring system that is composed of an indole and a nine-membered macrocyclic lactam (Figure [1]A).[33] [34] [35] Closely related natural products such as (–)-indolactam I (2)[36] have been isolated (Figure [1]A) as well as compounds that likely originate from the direct oxidation or functionalization of 1 (Figure [1]B).[37] [38] In addition, the majority of the indolactam alkaloids possess a terpene-derived motif at the C7 position of the indole heterocycle (Figure [1]C).[39] [40] [41] For example, (–)-pendolmycin (3)[42] maintains a C7-reverse-prenyl group, whereas (–)-7-geranylindolactam V (4)[43] maintains a linear terpene-based chain. In addition, (–)-lyngbyatoxin A (5)[44] and (–)-teleocidin A2 (6) have linalyl substituents with different configurations at the C19 chiral center.[45] Importantly, many of these C7-functionalized alkaloids are metabolically oxidized to yield unique natural products (Figure [1]D).[46] [47] [48] Other biosynthetic processes can further result in novel C7-substituents,[49] [50] including dimers of the indolactam scaffold (Figure [1]E).[37] Lastly, the most structural complex members of this family are represented by the tetracyclic teleocidin B1-4[51] and olivoretin[52] [53] [54] natural products that have a hydrophobic ring linking the C6 and C7 positions (Figure [1]F).

The activity of the indolactam alkaloids as cPKC and nPKC agonists may offer therapeutic opportunities in a range of diseases. In addition, many of these natural products have single-digit nanomolar affinity for PKC isozymes.[47] ^, [55] [56] [57] Despite this high level of potency, several issues have hindered the advancement of these potential therapeutics. First, low isolation yields of these alkaloids have led to an incomplete understanding of their respective biological profiles.[36,58] Second, the structural complexity of these alkaloids has slowed synthetic efforts to prepare the natural products. This limitation has affected structure-activity analyses of the scaffold and resulted in the use of lengthy synthetic routes or microbial methods to prepare analogues.[59–63] Third and most importantly, certain indolactam-based natural products have tumor-promoting behavior in particular skin-based cancer models.[64] This latter feature has also been established for various classes of PKC agonists (e.g., phorbol esters and aplysiatoxins), but recent studies have demonstrated that precisely constructed analogues can reverse these effects, yielding agents with antineoplastic properties.[25] [65] [66]

Our laboratory seeks to develop novel strategies to address both the synthetic and biological challenges associated with indolactam alkaloids. Our objectives are multifaceted: (1) provide an in-depth understanding of the structure-activity relationships present in the indolactam scaffold, (2) optimize the therapeutic potential of indolactam analogues to identify leads for pre-clinical advancement, and (3) demonstrate the efficacy of these agents in pre-clinical studies of PKC-dependent disease models. This review highlights our laboratory’s recent studies on the synthesis of indolactam-based natural products, the development of new strategies for scaffold diversification, and the biological evaluation of these alkaloids and analogues in cancer models.

 

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2

Total Syntheses of Indolactam Alkaloids

2.1

(–)-Indolactam V (1)

(–)-Indolactam V (1) retains the key pharmacophoric elements for interaction with the PKC C1 domain. In turn, numerous laboratories have devised synthetic routes to access this natural product. These strategies rely on a variety of innovative transformations including peptide coupling-based ring closure,[33] [34] ^, [67] [68] [69] [70] [71] [72] benzyne capture for carbon–heteroatom bond formation,[73] [74] photochemical methods,[75] and other approaches.[76] [77] Key challenges discovered in these previous syntheses included (1) promoting ring closure of the macrolactam and (2) the efficient installation of the N-arylvaline motif at the C4 position of the indole. Because 1 represents the core structure of the indolactam alkaloids, we sought to develop a modular approach that would be amenable to the large-scale synthesis of the natural product and further allow for the rapid preparation of analogues.

Figure 1 Representative natural products in the indolactam alkaloid family

In 2016, our laboratory reported an eight-step, stereoselective synthesis of 1 (Scheme [1]A).[78] Our approach commenced with the development of a copper-catalyzed method for the arylation of 4-bromoindole with l-valine to establish the key N-arylvaline motif. We initially discovered that various conditions promoted the coupling but resulted in undesired amine oxidation followed by hydrolysis.[79] The desired coupling product could be obtained under optimized conditions using an inert atmosphere and an appropriately protected indole 7. Gratifyingly, no epimerization of the amino acid α-center was observed. Importantly, this arylation protocol was effective for a wide range of amino acids, which may provide opportunities for scaffold diversification. For example, we found that an assortment of hydrophobic amino acids readily reacted in our system with yields exceeding 80%.[80]

Scheme 1 Synthesis of natural products (A) indolactam V and (B) indolactam I

Despite these initial advancements, the intermediate produced via the coupling of l-valine with N-tosylindole was found to degrade rapidly when exposed to air. Therefore, we developed a three-step procedure in which amino acid arylation, followed by reductive amination and peptide coupling with l-serine methyl ester, could be conducted in succession with only a single purification of the dipeptide product 8. To remove the indole protecting group, this intermediate was exposed to magnesium, which serendipitously also resulted in dehydration to form an acrylate 9.[78] Cyclization of this intermediate had been previously reported using ZrCl₄ as a Lewis acid catalyst.[73] In our hands, we were able to employ a similar protocol to form the nine-membered ring 10 and then obtain the desired primary alcohol of 1 through two further transformations.

This route provided 1 in eight steps. However, certain transformations in the sequence were sensitive to the reaction conditions and yielded lower amounts when employing multi-gram quantities. To address these issues, we performed a systematic optimization of the reactions at larger scales. These efforts led to updated protocols that allowed for the production of 1 in sufficient quantities for more advanced synthetic and medicinal chemistry studies.[81]

 

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2.2

(–)-Indolactam I (2)

(–)-Indolactam I (2) was first isolated from Streptoverticillium blastmyceticum in 1990. This natural product is structurally similar to 1, but it features an l-isoleucine subunit in place of the l-valine motif in 1.[36] Modifications at this position (C12) of the scaffold are known to play an important role in the potency of these compounds for PKC, as more hydrophobic C12 groups facilitate interaction with the cellular membrane during enzyme activation.[82] Despite the various synthetic routes to 1, methods for producing 2 and related C12 analogues are limited.

For example, at the time that we initiated these indolactam alkaloid-based research efforts, 2 had never been prepared by total synthesis and was only available in microgram quantities via microbial methods.[59]

Our recent application of a robust amino acid arylation protocol in the synthesis of 1 led us to apply this methodology in the preparation of 2, which could further offer opportunities for diversification of the C12 position in the scaffold (Scheme [1]B). In this approach, l-isoleucine smoothly underwent copper-catalyzed Ullmann coupling with 7. The resulting intermediate was again found to be sensitive to air exposure and thus was immediately subjected to reductive amination with formaldehyde, followed by peptide coupling with serine methyl ester. Indole deprotection and alcohol dehydration were simultaneously achieved using magnesium in methanol. Macrocyclization was accomplished with ZrCl₄, yielding a single stereocenter. The natural stereoconfiguration was achieved through epimerization using a weak base. Finally, ester reduction yielded the natural product 2 in eight steps with sufficient yield for thorough biological evaluation.[80]

 

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2.3

(–)-Pendolmycin (3)

Beyond natural products 1 and 2, the vast majority of indolactam alkaloids possess a substituent at the C7 position of the indole (Figure [1]C–F). For example, (–)-pendolmycin (3) incorporates a reverse-prenyl motif (Figure [1]C). Importantly, isoprenoid-derived groups at the C7 position are known to increase the potency of these natural products, as the hydrophobic substituent provides additional stabilization of the PKC-ligand-membrane complex during enzyme activation.[83] To better understand the structure-activity relationships afforded by C7 substituents, we sought to initially develop a protocol for the integration of terpene-based groups at this position and thereby prepare natural product 3.

The reverse-prenyl group in 3 presents a unique synthetic challenge because it is attached at a remote position on the indole heterocycle. As such, only two synthetic routes have been reported to 3. The first strategy was described by Natsume and co-workers in 1990, where the natural product was prepared in 16 steps.[84] However, the synthesis was plagued by multiple low-yielding transformations. More recently, Garg and co-workers disclosed a palladium-catalyzed α-arylation method that promoted the cross-coupling of an indolactam-derived heteroaryl halide with a zinc-stabilized amide enolate. This process led to successful formation of the C7-C19 carbon–carbon bond found in 3.[74] Despite the effectiveness of this transformation, multiple steps were still required to complete the synthesis, and ultimately 23 total steps were necessary to prepare the natural product.

To address the issues associated with these previous synthetic strategies, our laboratory sought to develop an approach in which 1 could be rapidly modified to yield the more structurally complex natural product 3 (Scheme [2]A). This objective led us to pursue a regioselective palladium-catalyzed Suzuki–Miyaura reaction that employed simple prenyl-derived organoboranes. The advantage of this strategy was that it provided the reverse-prenyl group of 3 without requiring further modifications of the C7-substituent post-cross-coupling. To execute this method, we adapted a protocol by Moreno and Kishi that allowed for selective bromination of the C7 position of the protected indolactam 12.[85]

Scheme 2 Synthesis of natural products (A) pendolmycin and (B) 7-geranylindolactam V, lyngbyatoxin, and teleocidin A-2

Using this electrophile 13, we next turned to the Suzuki–Miyaura reaction with a prenyl-based boronate ester. A key challenge for the execution of this method was the potential to generate two regioisomers, leading to either a prenyl or reverse-prenyl motif. We successfully accomplished this transformation by applying a catalyst system of [(allyl)PdCl]₂ and dicyclohexyl(2-(2-methoxynaphthalen-1-yl)phenyl)phosphine, which had previously been described by Yang and Buchwald for use on simple unsymmetrical allyl groups.[86] This transformation provided the desired carbon–carbon bond in a 46% yield with only the reverse-prenyl regioisomer observed.[87] Subsequent deprotection of the cross-coupling product yielded 3 in 12 total steps from commercially available starting materials, representing the shortest reported synthesis of the natural product.

 

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2.4

(–)-7-Geranylindolactam V (4), (–)-Lyngbyatoxin A (5), and (–)-Teleocidin A2 (6)

Similar to 3, several indolactam alkaloids incorporate terpene-derived chains at the C7 position (Figure [1]C). Natural product (–)-7-geranylindolactam V (4), for example, possesses a geranyl group. Although this alkaloid was first isolated and characterized in 1994 by Irie and co-workers, it had yet to be prepared by total synthesis prior to our studies.[43] Related natural products (–)-lyngbyatoxin A (5) and (–)-teleocidin A2 (6) have linalyl-based substituents at the C7 position but have different configurations at the C19 chiral center. Reports by Natsume[88] and Garg[74] led to total syntheses of 5 and 6. However, issues with stereocontrol, low-yielding transformations, and/or lengthy step-counts were encountered.

Our approach to natural products 4–6 was to implement a palladium-catalyzed Suzuki–Miyaura reaction similar to the one that had been successfully used to access 3 (Scheme [2]). This methodology offered an additional synthetic challenge because our objective was to demonstrate sufficient regiocontrol to prepare either geranyl-based 4 or linalyl-derivatives 5 and 6. Importantly, upon optimization of the Pd/ligand catalyst systems, this cross-coupling strategy could theoretically enable a single combination of a nucleophile (geranyl boronate ester) and an electrophile (13) to selectively yield the appropriate natural product in a regiodivergent manner (Scheme [2]B). To test this approach, we initially applied the previously successful catalyst system using geranyl boronate ester and 13 (Scheme [2]B). However, this nucleophile proved ineffective under these reaction conditions, and several attempted optimizations (e.g., adjusting temperature) led to only trace amounts of product. Fortunately, we discovered that employing Pd₂dba₃ as a precatalyst afforded carbon–carbon bond formation in yields ranging from 21–50% yield, depending on the Pd/ligand ratio. The regioselectivity of these reactions ranged from 76 to 80% (combined yield of linalyl-based products). The yield was improved to 56% with a regioselectivity of 87% under the optimized conditions. The linalyl-based coupling products underwent deprotection to yield 5 and 6. Each of these natural products was prepared in 12 steps, representing the shortest reported syntheses of these alkaloids.

We next turned to the synthesis of 4 with the goal of identifying a suitable catalyst system that could successfully reverse the regioselectivity of the Suzuki–Miyaura reaction to yield the geranyl motif (Scheme [2]B). ^tBuXPhos was initially used as the supporting ligand with an [(allyl)PdCl]₂ precatalyst, but only trace amounts of product were formed. Fortunately, we discovered that the Pd₂dba₃/XPhos catalyst system was highly active in the transformation, achieving a 77% yield while maintaining 77% regioselectivity for the desired geranyl motif. The coupling product from this reaction underwent deprotection to yield 4. This strategy enabled the first total synthesis of this natural product in only 12 steps.[89]

 

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3

Biological Studies of Indolactam Alkaloids

3.1

Pharmacophore of the Indolactam Scaffold

Natural product 1 was isolated in 1984 and identified as the core structure present in more complex alkaloids such as teleocidins A1-2 and B1-4 (Figure [1]C and 1F),[35] which had been previously discovered.[44] [90] Importantly, 1 was also found to be a potent agonist of PKC, and the pharmacophore was subsequently determined to include several components of the macrolactam: the carbonyl of the amide, the NH of the amide, and the primary alcohol appendage at the C9 position (Figure [2]).[57] The latter feature is of particular note because studies have demonstrated that further functionalization of the alcohol leads to a loss of potency for PKC isozymes.[91] [92]

Figure 2 Structure-activity relationships of indolactam scaffold

Early studies suggested that the natural (9S,12S) configurations of the two chiral centers in 1 were ideal for PKC binding. Specifically, Fujiki and co-workers showed (9S,12R)-(+)-indolactam V (16) and (9R,12R)-(–)-indolactam V (17) were inactive in HL-60 leukemia cell adhesion assays, whereas (9R,12S)-(+)-indolactam V (18) retained activity, albeit significantly decreased (Scheme [3]).[93] Interestingly, more modern studies have shown that 17 has increased neuroprotective effects and decreased neurotoxicity compared to 1.[94]

Scheme 3 Synthesis of stereoisomers of indolactam V

In order to clarify the biological profiles of 16–18, we sought to utilize our modular synthetic route to prepare these compounds more efficiently than described in previous reports. Subsequent cell-based assays and computational studies would also allow us to identify the stereochemical requirements for PKC-mediated activity. To this end, we adapted our synthesis to 1 in order to produce 16–18 (Scheme [3]). Stereoisomer 18 could be produced readily by omitting the final C9-epimerzation step in our original route. Instead, the direct reduction of intermediate 11 provided stereoisomer 18 in seven total steps. The routes to 16 and 17 required modifying the stereochemistry of the amino acid initially employed in the second step of our original synthetic route. Thus, in this approach, d-valine was arylated through a copper-catalyzed reaction with 7. Subsequent reductive amination and EDC-catalyzed peptide coupling produced dipeptide intermediate, which reacted with magnesium to afford the acrylate. ZrCl₄-mediated ring closure yielded the macrocycle 15, and this reaction proved to be highly stereoselective for the newly formed C9 stereocenter. The ester 15 could then be directly reduced to yield stereoisomer 16 in only seven steps. Alternatively, 15 could undergo C9-epimerization with a weak base, followed by ester reduction to produce stereoisomer 17 in eight steps.

Cellular assays and modeling studies were performed with all stereoisomers of 1. In vitro experiments examined growth inhibition in two cancer cell lines: K562 (chronic myelogenous leukemia) and U937 (histiocytic lymphoma). Bryostatin 1, a PKC C1 domain agonist that has entered clinical trials, was also evaluated for comparison. Natural product 1 demonstrated greater activity relative to bryostatin 1 in the K562 cell line, whereas both compounds exhibited statistically comparable effects in U937 cells. Importantly, stereoisomers 16–18 proved to be inactive in both cell lines. To rationalize these results, molecular docking studies were performed with 1 and 16–18 using the PKCδ C1B domain.[81] All stereoisomers were found to adopt a cis-amide (twist) conformation, and, as previously reported, 1 exhibited similar hydrogen-bonding interactions with key amino acids (Thr-242, Leu-251, and Gly-252).[95] Additionally, the indole ring was sufficiently close to the hydrogen atom of Pro-241 (2.64 Å), allowing for a stabilizing CH/π interaction. Stereoisomers 16 and 18 each result in a loss of at least one of these key interactions, and no energetically favorable binding conformation was discovered for 17. Collectively, these results suggest that (1) the natural configurations of 1 are necessary to promote high affinity for the cPKC and nPKC isozymes and (2) this feature can be rationalized through non-ideal interactions of these ligands in the C1 domain active site.[81]

 

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3.2

Non-Pharmacophore Considerations for the Indolactam Scaffold

The endogenous ligand DAG plays dual roles during PKC activation. The ligand pharmacophore binds to the PKC C1 domain and the hydrophobic alkyl chains of the DAG directly interact with the membrane. Thus, DAG stabilizes the PKC-DAG-membrane complex and anchors the protein to the membrane in order to activate the enzyme.[96] Similarly, various positions (C6, C7, and C12) of the indolactam scaffold have been proposed to engage the cell membrane (Figure [2]), and previous studies have demonstrated that hydrophobic groups at these positions can modulate the binding affinity of indolactam analogues to PKC C1 domains.[97] [98]

In previous studies, we sought to examine the functional properties of indolactam natural products with various substitution patterns at key positions of ligand-membrane association. Therefore, we performed a comparison of alkaloids 1, 2, and 5 using cell growth inhibition assays in the human chronic myelogenous leukemia cell line K562. Natural products 1 and 2 vary in the R-group (isopropyl vs. sec-butyl, respectively) at the C12 position, and both were found to be equally potent in this assay. However, alkaloid 5, which possesses a linalyl group at the C7 position, was discovered to have a statistically significant decrease in activity. Importantly, these results oppose the generally accepted correlation that increased hydrophobicity at key positions (e.g., C6, C7, or C12) necessarily leads to higher potency. Instead, although the nature of the membrane-interacting groups on the indolactam analogues can have practical effects on PKC signaling, these agents still must be evaluated in appropriate disease models to demonstrate their therapeutic applications.[80]

 

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3.3

Indolactams as Tumor Growth Suppressors

Early animal studies with phorbol esters found that the co-application of these natural products with a carcinogen led to accelerated tumor growth on the skin of mice.[99] Subsequently, Blumberg and co-workers discovered that phorbol esters are potent agonists of the PKC C1 domain.[100] These investigations led to the classification of phorbol esters as ‘tumor promoters’, which induce undesired proliferative effects in cancer cells via activation of cPKC and/or nPKC isozymes. Although a variety of PKC C1 domain agonists have traditionally been labeled as tumor promoters, an important exception is the polyketide natural product bryostatin 1. Contrary to phorbol esters, bryostatin 1 exhibits antiproliferative effects in carcinogen-based skin tests.[101] In addition, this ‘tumor suppressor’ natural product has consistently demonstrated anticancer activity in a range of preclinical models.[102] In turn, bryostatin 1 has been subjected to >30 clinical trials for multiple cancers, Alzheimer’s disease, and HIV latency.[103] Despite this clinical significance, bryostatin 1 suffers from a low isolation yield from its source Bugula neritina.

In addition, the structural complexity of the natural product presents unique synthetic challenges and to date has limited the pharmaceutical advancement of this potential therapeutic.[66]

Given the supply problem with bryostatin 1, a critical objective in this field is to identify other PKC C1 domain agonists that display tumor suppressive activity. In recent years, several insights have led to progress towards achieving this goal. First, Newton and co-workers performed comprehensive studies demonstrating that loss-of-function mutations in PKC isozymes constitute a primary mechanism through which cancer cells halt the protective effects of these enzymes.[16] Importantly, these results suggest that the application of small-molecule PKC activators is a more promising chemotherapeutic strategy than PKC inhibitors, which have yet to make a clinical impact despite decades of trials.[104] In addition to these insights, researchers have discovered that many natural products classified as tumor promoters actually deplete intracellular PKC. For example, phorbol esters such as phorbol 12-myristate 13-acetate (PMA) are highly potent agonists that induce rapid activation of cPKCs and nPKCs, but prolonged signaling ultimately leads to pan-degradation of these enzymes.[105] [106] This latter event results in the silencing of PKC signaling and thus an inability of the cancer cell to initiate apoptotic pathways. Therefore, the tumor-promoting behavior of natural products such as phorbol esters originates from the hyperactivation and subsequent loss of PKCs.[31] Importantly, researchers have found that precise structural modifications to phorbol esters and other tumor promoters can mitigate these effects and lead to compounds with tumor-suppressive properties. The most notable examples are the phorbol ester prostratin[107] and the simplified aplysiatoxin analogue aplog-1.[108] [109] In both instances, the removal of hydrophobic, membrane-interacting groups resulted in the reversal of activity, producing compounds with anticancer profiles similar to that of bryostatin 1. Inspired by these revelations in the PKC field, our laboratory sought to explore whether the indolactam alkaloids – many of which have historically been classified as tumor promoters – could be systematically modified to control their tumor-suppressive versus -promoting behavior. A key advantage of employing indolactams, relative to other PKC C1-targeting natural products, is that the scaffold is rapidly accessible by our synthetic routes and thus could provide a more viable platform for pharmaceutical development.

To pursue these investigations, we required suitable methods for predicting the anticancer properties of novel indolactam analogues.

In 2011, Blumberg and co-workers reported a panel of in vitro assays that could be used to effectively classify new PKC C1 domain ligands as either tumor promoters such as PMA or tumor suppressors such as bryostatin 1. The cell-based assessments included growth inhibition experiments in the human prostate carcinoma cell line LNCaP. In addition, cell attachment and growth inhibition assays were performed in the human lymphoma cell line U937. Tumor promoters characteristically exhibit high levels of growth inhibition in both cancer cell lines and, importantly, a significant degree of cell attachment in U937.[110]

In our studies, we examined whether specific structural modifications to the indolactam scaffold could alter the tumor suppression versus promotion behavior of these compounds. Initially, a set of indolactam analogues was synthesized through direct derivatization of the 1 (Figure [3]A). First, the indole nitrogen (N1 position) was either alkylated or acylated via a facile three-step sequence.[87] Since lipophilicity has historically been a metric for predicting tumor suppression versus promotion activity,[111] we appended functional groups at the N1 position that varied in hydrophobicity, size, and electronic properties to produce 19–22.[82] [87] In addition to these analogues, strategies were developed to functionalize the C7 position of 1. This approach led to the preparation of 23 and 24, although the latter proved to be unstable upon isolation. These synthetic compounds were combined with indolactam natural products and previous analogues to afford a library for testing in tumor suppression assays.

Figure 3 (A) Synthesis of indolactam analogues. (B) Induction of cell attachment in U937 cells upon treatment with PKC activator bryostatin 1, PMA, or representative indolactams. Cells were treated with four concentrations (1, 10, 100, and 1000 nM; left to right columns) of test compound for 72 h. Attached cells values are expressed as a percentage of the total cell number at the end of the assay. All values are the mean of n > 3 biological replicates ± s.d.

This focused library was assessed using the previously reported assay panel for the in vitro evaluation of tumor suppression versus promotion behavior (Figure [3]B). Bryostatin 1 and PMA were used as controls for the study. As previously reported, parent structure 1 displayed tumor promotion behavior that resembled that of PMA. This activity was also discovered in other natural products (e.g., 3 and 5) with a clear trend indicating that more lipophilic motifs result in higher levels of tumor promotion behavior. Importantly, this activity could be reversed through suitable indole substitution at the N1 position. Specifically, indolactam analogues 20 and 22 demonstrated tumor suppression effects in all assays, resembling the activity of bryostatin 1. These compounds feature groups at the N1 position with different electronic properties but possess relatively similar steric environments. Interestingly, both 20 and 22 display selectivity for nPKC isozymes relative to cPKCs.[82] Collectively, this study demonstrated that suitable derivatization of the indolactam scaffold could enable opportunities to regulate tumor suppression versus promotion properties and thereby offer novel chemotherapeutic opportunities.[112]

 

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3.4

Indolactams as Gli Inhibitors

Another principal objective of our research program is to identify new connections between PKC biology and human health. Dysregulated PKC signaling is a feature of numerous diseases,[17] but the relationship between PKC isozymes and their deleterious cellular effects often remains ambiguous. These factors create challenges in terms of (1) assigning mechanistic roles for specific PKC isozymes in a disease state and (2) the strategic development and clinical application of PKC-targeting therapeutics.[104] To advance this field, our laboratory has created a PKC effector library containing compounds that possess structural diversity, different modes of action, and unique PKC isozyme/class selectivities. This platform allows for an unbiased evaluation of PKC-mediated effects in a disease model and offers insights into the optimal method for targeting PKC in drug design.

We recently demonstrated our approach in a collaborative project examining the Hedgehog (Hh) signaling pathway, which is a driving factor in several major human cancers including many lethal medulloblastomas and basal cell carcinoma. This undesired effect of Hh signaling is due to the activation of oncogenic Gli transcription factors.[113] Since Hh pathway activity is known to be regulated by PKC isozymes,[114] [115] we conducted a screen of diverse PKC effectors to identify molecules that can act as Gli inhibitors (Figure [4]). We discovered that both natural product 1 and a benzolactam-derived analogue of indolactam V (TPPB) were highly potent inhibitors of Gli in multiple Hh-dependent cellular models including fibroblast-derived Shh-LIGHT2, C3H10T1/2 mesenchymal stem cells, and the ASZ001 basal cell carcinoma line. Importantly, these PKC-targeting compounds remained active at nanomolar concentrations even in a drug-resistant cell line Sufu-KO-LIGHT, in which all currently clinically relevant Gli inhibitors are ineffective.[116] These studies demonstrate that our platform can be used to identify new conditions in which PKC is an actionable target for treatment. Importantly, this investigation led to the discovery of a novel therapeutic application of indolactam alkaloids and related analogues in Hh-dependent cancers.[87]

Figure 4 (A) Structure of FDA-approved vismodegib (GDC) and indolactam analogue TPPB. (B) Relative Gli-driven luciferase activity at 1.5 μM dose of GDC, TPPB, or 1 in Shh-LIGHT2 and Sufu-KO-LIGHT cells. Gli luciferase is calculated relative to Gli-driven luciferase activity induced by 200 nM SAG in Shh-LIGH2 cells (100%) or DMSO in Sufu-KO-LIGHT cells (100%). All values are the mean of n > 3 biological replicates ± s.d.

To provide further inroads into the development of Gli inhibitors, we examined a range of indolactam alkaloids and analogues using in vitro models of Hh signaling. These studies incorporated indolactam natural products (1–3 and 5), N1-substituted derivatives (19–22), C7-functionalized compounds, and further analogues 23–24. These experiments revealed that 21, which possesses a hydrophobic hexyl group at the N1 position, outperformed all evaluated analogues. In addition, 21 proved to have enhanced potency for Gli inhibition relative to our initial hit, natural product 1, in a range of cellular models of Hh signaling. Collectively, these studies demonstrate new therapeutic opportunities for the application of PKC-targeting indolactams to combat Gli-promoted cancers.[87]

 

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4

Conclusions and Outlook

Despite the first isolation of an indolactam alkaloid over 60 years ago, this family of natural products remains intriguing for both synthesis- and therapeutic-driven investigations. As described in this review, our laboratory has made significant contributions in terms of the synthesis of the core indolactam natural products (1–2) as well as terpene-derived alkaloids (3–6). From these efforts, we have been able to expand the scope of indolactam-based analogues that can be rapidly produced for biological testing. Beyond our studies, recent work by Baran and co-workers has provided an innovative approach to efficiently prepare teleocidin B1-4, which possesses a tetracyclic structure.[77] Still, the majority of family members have yet to succumb to total synthesis efforts. In addition, remote positions (i.e., C5 and C6) of the indole heterocycle remain challenging to modify synthetically, as seen by efforts to prepare natural products and analogues with these substitution patterns.[77] [95] [117] [118] Therefore, despite the likely importance of these positions for ligand-membrane interactions, only a limited number of such analogues have been prepared or biologically evaluated. Future synthetic studies of the indolactam alkaloids should adopt strategies that allow for the preparation of a wider range of family members, while still incorporating methodologies that provide the means for rapid diversification.

The recent insights into the tumor suppressive functions of PKC enzymes have established new therapeutic opportunities using PKC activators such as the indolactam alkaloids. Our laboratory has described structure-activity relationships for the indolactam scaffold, which in turn can inform drug design strategies. For example, we have identified the need for the natural stereoconfigurations at C9 and C12 positions as well as the role of hydrophobic groups at C6, C7, and C12 for maintaining membrane association during PKC activation. Importantly, we have also pinpointed key positions (i.e., the indole N1 substituent) that can be modified to enhance the tumor suppressive properties of indolactam analogues. Moreover, we have discovered a previously unknown activity of indolactams as Gli inhibitors with potential application in Hh-dependent cancers. A promising future direction in this field is the examination of next-generation indolactam alkaloids, which have been optimized for therapy, in more advanced preclinical cancer models to assess their viability as chemotherapeutics.

 

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Conflict of Interest

The authors declare no conflict of interest.

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Publication History

Received: 26 February 2025

Accepted after revision: 21 March 2025

Accepted Manuscript online:
21 March 2025

Article published online:
29 April 2025