Growth inhibition values (GI50) of NCI’s human tumor cell line panel expressed in log scale.
SHetA2 (NSC 721689) is a novel synthetic flexible heteroarotinoid that has promising cancer-preventive activity, and has exhibited growth inhibition on 60 cancer cell lines in vitro, along with ovarian, lung, and kidney cancers in vivo. It binds and interferes with the function of a molecular chaperone, mortalin, leading to mitochondrial swelling and mitophagy that induce apoptosis in cancer cells without harming normal cells. It showed minimal toxicity in preclinical studies and thus is now in Phase-0 clinical trial. This chapter summarizes its evolution, synthesis, structure-activity relationship, mechanism of action, pharmacokinetics, and potential clinical applications in last 12 years. It also provides insights into designing more potent and safer SHetA2 analogs for future cancer-preventive drug development.
- flexible heteroarotinoids
- cancer prevention
- ovarian cancer
Cancer-preventive agents are biologics, dietary supplements, nutrients or marketed drugs used to reduce the risk of developing cancer or prevent the recurrence of cancer . The cancer-preventive actions of these agents are mediated by a variety of proposed mechanisms, which include inhibition of oxidative and inflammatory stress, immunomodulatory action, induction of cell differentiation and apoptosis, and inhibition of cell growth and angiogenesis. Hundreds of natural and synthetic compounds have been shown to inhibit cancer cell growth including vitamins (vitamins A, D, and E), organosulfur compounds (brassinin, sulforaphane, and isothiocyanates), minerals (calcium and selenium), phytoestrogens (resveratrol), flavonoids (genistein and quercetin), synthetic compounds (retinoids, sulforamate, and prenylated flavonoid analogs) , and marketed drugs (tamoxifen, raloxifene, finasteride, aspirin, sulindac, difluoromethylornithine, and metformin) . Drug development for cancer prevention imposes distinct challenges beyond those associated with drug development for cancer therapy. One of the challenges is that cancer preventive agents must have higher therapeutic index. Nevertheless, the potential to intervene prior to the cancer developing is an attractive strategy to fight cancers .
A novel class of anticancer agents that has demonstrated such qualities is the flexible heteroarotinoids (Flex-Hets). Flex-Hets, derived from the retinoids, have exhibited potential anticancer activities in various cancer cell lines, while displaying minimal toxicity to normal cells. Among these compounds, SHetA2 [(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl]amino]methane-thione) exhibited the greatest growth inhibitory efficacy and potency against various cancer cell lines and was chosen as a lead compound for further development [5–7]. SHetA2 has been shown to interfere with mortalin binding to p53 and p66 Src homologous-collagen homologue (p66shc) leading to apoptosis in cancer cells . Furthermore, it has been shown to induce both intrinsic  and extrinsic apoptotic pathway , cause cell cycle arrest , induce differentiation , and inhibit angiogenesis  in cancer cells, while displaying negligible toxicity in animal models . Hence, SHetA2 was regarded as a novel class of promising anticancer agent that selectively targeted the cancer cells. Consequently, it was evaluated in preclinical development for cancer prevention through the National Cancer Institute (NCI) Rapid Access to Intervention Development (RAID) and Rapid Access to Preventive Intervention Development (RAPID) program . Studies in rats and dogs showed that no toxicity was observed in any of the tested dosage groups. The no-observed-adverse-effect-level (NOAEL) for SHetA2 was not established and was considered to be above 1500 mg/kg/day in dogs . The therapeutic window for administrative safety with SHetA2 was determined to be 25- to 150-folds above
2. History of retinoid development
Retinoids, including natural retinoic acid (RA) and its synthetic derivatives, are a group of promising anticancer agents that have shown both chemotherapeutic and chemopreventive potential in both animals and humans [15–17]. They exhibit therapeutic properties by activating the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) . Each receptor has three subtypes (-α, -β, -γ), and they belong to a family of proteins that regulate the transcription of genes responsible for a variety of metabolic, developmental, and differentiation pathways in cells and tissues . Retinoids have been shown to inhibit growth, and induce differentiation and apoptosis of cancer cells both
Recognizing the potential chemopreventive and chemotherapeutic effects of retinoids, chemists have tried to design and synthesize new retinoid derivatives that express high cytotoxic potential with lower toxicity. The rationale is that by developing compounds that selectively activate only one retinoid receptor subtype, the associated toxicities could be reduced. For example, RXR-selective compounds were thought to have great potential for pharmacological use against tumors and other diseases due to a large number of ligand activated receptors whose activities involve dimerization with RXR receptors . Bexarotene (Targretin®, with a one-carbon linker), is a selective RXR agonist and the first synthetic arotinoid approved for the treatment of all stages of cutaneous T-cell lymphoma . RARγ, selectively expressed at high levels in the skin, has been shown to be the mediator of retinoid activity . Early efforts to increase the therapeutic ratio of retinoids included increasing the structure rigidity by incorporating a double-bond between the two aromatic rings, leading to the development of arotinoids with two atom linker. For example, TTNPB (Figure 1, 5), an arotinoid with significant anti-tumor activity  was found to be selective for RAR receptors . Unfortunately, intolerable toxicities observed in animal models limited its clinical utility .
Subsequent structural modifications involved the addition of one heteroatom (O, N, and S) in the cyclic ring of the arotinoids to block its oxidation into toxic metabolites. This resulted in the development of a new class of compounds called the heteroarotinoids (Hets, Figure 1, 6–12). These Hets exhibited similar biological activities to retinoids, but with significantly reduced toxicities [29, 31].
2.4. Flexible heteroarotinoids
Different linkers were placed between the two aryl groups of the Hets to increase their rigidity, providing a more specific fit into each receptor’s binding pocket. Two-atom linkers, such as amide (Figure 1, 8) and esters (Figure 1, 9–11) were synthesized, and they showed varying degree of receptor selectivity. Interestingly, Hets with either the three-atom urea (Figure 1, 14, 17, 18) or thiourea (Figure 1, 15, 16, 19, 20) linker demonstrated significant anticancer activity without activating any of the retinoid receptors . These compounds showed significant growth inhibitory activities against the ovarian cancer cell lines: Caov-3, OVCAR-3, and SK-OV-3, while exhibiting low activities against normal and benign cells. Moreover, due to the lack of RAR/RXR activation, these compounds did not exhibit the associated toxicities observed with other retinoids. Among these compounds, SHetA2 (Figure 1, 16) demonstrated the greatest potency against the aforementioned ovarian cancer cell lines at concentrations ranging from 0.2–3.7 μM. Since both urea and thiourea linkers are somewhat flexible in nature, these compounds were termed flexible heteroarotinoids (Flex-Hets). The evolution of SHetA2 is shown in Figure 2.
3. SHetA2, a Flex-Het
Flex-Hets have shown cancer-preventive activity by regulating apoptosis, cell growth, and differentiation in multiple types of cancer cell lines. They display significantly greater growth inhibition activities against ovarian cancer cells than both normal and benign ovarian cells . Among the Flex-Hets, SHetA2 has shown to induce the highest levels of apoptosis in multiple cancer cell types, while retaining the differential resistance in normal cells . SHetA2 functions independently of retinoid receptors  and therefore, lacks the toxicities associated with conventional retinoids. More importantly, SHetA2 differentially induces apoptosis in cancer cells while sparing normal cells . All 60 cancer cell lines in NCI human tumor panel were sensitive to the growth inhibition activities of SHetA2 at micromolar concentrations  (Table 1). Despite this wide range of activity against cancer cells, normal and benign human ovarian, endometrial, and oral cultures were resistant to SHetA2-induced apoptosis and growth inhibition [6, 9]. In a recent study, SHetA2 was tested to be negative across three genetic toxicology assays, namely the
|Cancer panel||Cell line||Log GI50 (SHetA2)||Log GI50 (ATRA)|
|Nonsmall-cell lung carcinoma||A549/ATCC||−4.9||−4.3|
|Colon cancer||COLO 205||−4.9||−4.2|
4. Synthesis of SHetA2
The synthesis of SHetA2 mainly involved the preparation of the intermediate, aminothiochroman (Figures 3 and 4). Early synthetic procedure (Figure 3) involved using Fe/HOAc to reduce the nitro group with a yield of 40% . The nitration reaction was the problematic step with only a 26% yield, since many additional by-products were formed in this reaction. In order to address this issue, Tallent et al. employed a nitrogen-containing starting material (4-acetamidobenzenethiol)  (Figure 4). They used methyllithium instead of methylmagnesium bromide to form the intermediate carbinol. In the cyclization procedure to form the thiochroman, chlorobenzene was used as solvent with a 58% yield avoiding the use of flammable and noxious CS2. This method circumvented the low-yielding nitration step and the subsequent reduction step to generate the amine group of aminothiochroman. The improved synthesis was shorter and afforded a 5-fold higher overall yield. The final product, SHetA2, was formed by reacting the aminothiochroman with 4-nitrophenyl isothiocyanate in tetrahydrofuran (Figure 4).
5. Structure-activity relationship
Structure-activity relationship (SAR) of SHetA2 and its derivatives has been valuable to identify important structural modifications that have contributed to its anticancer potency and selectivity, and to guide the design of more potent and less toxic SHetA2 analogs. Figure 2 illustrates the structural evolution of SHetA2 from ATRA. The goal was to increase the selectivity of retinoids towards RAR/RXR subtypes, so as to reduce the associated toxicities while retaining its anticancer activities. It has been shown that RAR-specific ligands can rescue
In order to reduce toxicity, a benzylic carbon in the tetrahydronaphthalene was replaced with a heteroatom (O, N, and S). The purpose was to prevent benzylic (metabolic) oxidation which could result in toxic metabolites. This single modification resulted in Hets (Figure 1, 6–12) with similar biological activities to RA but significantly reduced toxicities [29, 31]. An example was the diaryl heteroarotinoid (Figure 1, 6), a RAR-selective retinoid derivative. It differed from TTNPB by an oxygen heteroatom, but exhibited a significant decrease in toxicity, increasing the maximum tolerated dose (MTD) by 3000-fold as compared to TTNPB (Figure 1, 5) . Other monoaryl heteroarotinods (Figure 1, 7a, 7b) were also evaluated, and revealed a 3-fold decrease in toxicity along with a decreased ability to activate the RAR receptors when compared to ATRA (Figure 5) [40, 30].
5.1. Thiochroman rings
SAR studies have identified structures containing six-membered ring (Figure 1, 7a, 7b) tend to confer increased RARβ selectivity over five-membered ring systems (Figure 1, 12a, 12b), while sulfur heteroatom confers a greater RARγ selectivity over oxygen atom . The thiochroman ring system is flexible and has been shown to induce apoptosis to a greater extent than a rigid planar quinoline unit . These findings highlight the important role played by the thiochroman ring in enhancing the activity of Flex-Hets. Therefore, the thiochroman ring forms one of the fundamental moieties of SHetA2 and its analogs (14–20).
5.2. Two-atom linkers
To further increase the selectivity for each RAR and RXR receptors, various linkers were placed between the two aryl groups of the Hets by modifying their structure rigidity. Two-atom linker compounds such as amide (Figure 1, 8) and ester (Figure 1, 9) were reported [42, 43]. Compound (8) was found to be a receptor panagonist, while compound (9) was RXR selective. Both showed significant growth inhibitory activities against head and neck cancer using a tumor xenograft mouse model ; however, only compound (8) induced apoptosis in ovarian cancer cells. This indicated that RXR activation is sufficient to inhibit tumor growth, while activation of both RAR and RXR are required for the maximum activity, at the expense of toxicity. Other two ester-linked compounds (Figure 1, 10, 11) were found to activate both RARs and RXRs . On the other hand, these ester-linked Hets appeared to only induce growth inhibition but not apoptosis.
5.3. Three-atom linkers
Three-atom linkers have also been reported in the literature (Figure 4, 21, 22). It was suggested that the presence of a three-atom linker may increase RAR selectivity , and the ability for linkers to form hydrogen bonds may enhance RARγ selectivity . As such, a series of urea/thiourea derivatives were synthesized and evaluated (Figure 1, 14–20) [6, 7]. Unlike conventional retinoids, these Flex-Hets were able to induce selective and potent apoptotic activity in cancer cells independent of RAR/RXR activation . The only retinoid activity retained by Flex-Hets is the ability to induce differentiation and reverse the cancerous phenotype.
The X-ray crystal structure of the Flex-Hets showed that a unique lattice network formed through extensive intermolecular H-bonding between the NH and the oxygen of the highly polarized C=O urea linker in another molecule, whereas this was not observed in the C=S thiourea derivatives. It suggested that the urea derivatives may be more active than its thiourea counterpart. This observation is also supported by their
Regardless, the fact remains that millions of dollars have been invested by the NCI RAID and RAPID programs in the preclinical development of SHetA2, and many studies involving animal models have shown that SHetA2 is a potent and selective inducer of apoptosis with no significant toxicities. While SHetC2 lacks these extensive studies, it does show potential as the next chemopreventive drug candidate following SHetA2. These results indicate that the inclusion of three-atom urea/thiourea linker is critical to induce potent anticancer activities independent of RAR/RXR activation as observed in these Flex-Hets. Tables 2 and 3 summarize the effects of the structural modifications on the growth inhibition by these derivatives against various cancer cell lines reported to-date.
Substitutions on the phenyl group have also been evaluated . The nitro (NO2) substitution (Table 3, 16, 17) consistently exhibited greater growth inhibitory and apoptotic activity than their methyl (Table 3, 18–20) or ethyl ester counterparts (Table 3, 14, 15). This suggests that the nitro substitution may have enhanced the overall activity of the compound. Collectively, for the Flex-Hets, the thiochroman ring and nitro substitution are important for enhancing the anticancer activity, while the thiourea linker is crucial for RAR/RXR independent and selective anticancer activities against cancer cells.
5.4. Three-atom linker with thiochroman ring replaced
In order to expand the potential clinical applications, we designed, synthesized, and evaluated nine
5.5. Four-atom linker
Another approach to the structure modification of SHetA2 was to keep the thiochroman ring but replace the thiourea linker with a 4-atom acrylamide linker NC(O)C=C and various substitutions on the terminal aryl ring. When evaluated in a cytotoxicity assay of the human A2780 ovarian cancer cell line, results indicate that activity of 4-nitro phenyl analogs are comparable to that of SHetA2 with efficacies slightly reduced compared to SHetA2 . The α,β-unsaturated acrylamide may contribute to its increase in potency and the compound 24 (Figure 6) is the example.
6. Mechanism of action
The biological targets and SHetA2’s mechanism of action have been extensively studied. Coimmunoprecipitation experiments demonstrated that SHetA2 interfered with the binding of mortalin, a molecular chaperone to p53 and p66 Src homologous-collagen homolog (p66shc) in A2780 ovarian cancer cell line, leading to mitochondrial swelling and mitophagy and finally apoptosis . SHetA2 is also involved in modulating other cellular processes including cell-growth, differentiation, and angiogenesis.
6.1. Induction of apoptosis
6.1.1. Intrinsic pathway
SHetA2 is shown to selectively induce apoptosis in various cancer cell lines by targeting the mitochondria . Exposure of SHetA2 to squamous carcinoma cells resulted in a decrease in mitochondrial permeability transition, followed by the release of cytochrome
6.1.2. Extrinsic pathway (death receptor pathway)
Exposure of non-small cell lung cancer cells (NSCLC) to SHetA2 revealed the induction of the extrinsic apoptotic pathway involving the death receptor 5 (DR5) . It was shown that SHetA2-enhanced DR5 expression through the enhanced binding of CAAT/enhancer-binding protein homologous protein (CHOP) to its binding site located in the 5’-flanking region of the DR5 gene. Since CHOP is highly inducible during endoplasmic reticulum (ER) stress, this finding suggests that SHetA2 may act as an inducer of ER stress. The induction of DR5 expression leads to caspase 8-dependent apoptosis. Moreover, the induction of DR5 was shown to enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis (Figure 7). Further studies found that down-regulation of cellular FLICE-inhibitory protein (c-FLIP), and to a lesser extent of survivin, were involved in SHetA2-induced apoptosis, as well as enhancement of TRAIL-initiated apoptosis . c-FLIP is a major inhibitor of the extrinsic apoptotic pathway , while survivin modulates both intrinsic and extrinsic apoptotic pathways .
TRAIL and its receptors have also attracted much attention recently because TRAIL preferentially induces apoptosis in malignant cells while sparing most normal cells . SHetA2 was found to sensitize ovarian carcinomas that are resistant to these DR ligands, without harming the normal cells [54, 55]. This suggests a possible synergistic effect Further investigation identified that downregulation of NF-kB transactivation activity by SHetA2 was involved in this sensitizing effect, triggering extrinsic apoptosis in cancer cells resistant to certain chemotherapeutics . SHetA2 was shown to repress the upstream IkB kinase (IKK) activity that resulted in NF-kB downregulation. Apart from DR ligands, SHetA2 has also shown its chemosensitizing effects against cisplatin-resistant ovarian cancer through p53-independent pathways [6, 56]. However, such enhancement of sensitivities was not observed in resistant uterine cancer cell lines for a number of chemotherapeutics in combination with SHetA2 treatment (Figure 8) .
|Apoptotic pathways affected by SHetA2|
|Possible biological targets||Effect||Reference|
|Intrinsic||↓ GSH||Redox regulation|||
|↑ Bip/GRP78, IRE1α, ATF4, XBP1||ER stress|||
|↑ DR-5||Cell death|||
|↑ CHOP||Cell death|||
|↓ IKK||Cell survival|||
|↓ NF-kB||Cell survival|||
|Cell cycle pathways|
|↓ Cyclin D1||G1 arrest|||
|↓ AP-1||Cell-cycle progression|||
|Differentiation induction pathways|
|↑ Thrombospondin (TSP-4)||Antiangiogenic|||
|↓ Thymidine Phosphorylase (TP)||Angiogenic|||
|Signal transduction pathways|
|↓ KIT kinase (c-KIT)||Cell death|||
6.1.3. ROS and GSH
Increased reactive oxygen species (ROS) level was initially thought to be responsible for SHetA2-induced apoptosis. Multiple studies have documented the ROS generation along with mitochondrial swelling and the release of cytochrome
6.2. Induction of cell-cycle arrest and differentiation
Apart from apoptosis, SHetA2 repression of NF-kB expression also initiates a series of events that can lead to cell-cycle arrest and cell differentiation. SHetA2-induced Cyclin D1 degradation in both Caki-1 renal cancer and normal HK-2 cell lines results in the accumulation of cells in the G0-G1 phase . Cyclin D1 degradation alone has shown to be sufficient in inducing G1 cell cycle arrest . Cyclin D1 transcription can be induced by NF-kB through multiple NF-kB-binding sites in the Cyclin D1 promoter, which is consistent with the observed down regulation of Cyclin D1. Also, SHetA2 can induce cellular differentiation in kidney , as well as in ovarian cancer organotypic cultures and xenografts . Treatment of these cancer cells shows that 1 μM SHetA2 is sufficient to reverse the cancerous phenotype depending on the status of the cells. However, at higher concentrations of SHetA2, apoptosis would dominate over differentiation. The mechanism for differentiation induction is likely to be associated with the upregulation of E-Cadherin in renal cancer cells observed with the repression of NF-kB . Decrease or loss of nuclear E-Cadherin expression is associated with poor prognosis in kidney cancer, as it confers the ability to migrate and invade . Therefore, up-regulation or restoration of E-Cadherin’s function has been one of the therapeutic goals for anticancer treatments.
6.3. Antiangiogenic activity
Microarray analysis have suggested that SHetA2 treatment results in significant up-regulation of thrombospondin-4 (TSP-4), and down-regulation of thymidine phosphorylase (TP) in A2780 ovarian cancer cells. TSP-4 and TP protein levels followed those of the mRNA. TSP-4 was hypothesized to be antiangiogenic due to the presence of type-III repeats which is also found in TSP-1. The type-III repeats in TSP-1 has been reported to inhibit the binding of fibroblast growth factor-2 to endothelial cells, a process that leads to endothelial cell proliferation
6.4. Kinase inhibitory activity
SHetA2 is also being evaluated for its ability to inhibit kinase activity based on its structural similarity to sorafenib and linifanib (Figure 9) . Sorafenib is an FDA approved diarylurea multikinase inhibitor that inhibits tumor growth, while linifanib is a KIT-3 kinase inhibitor [66, 67]. All three compounds consist of a three atom urea or thiourea linker between two aromatic rings, of which this structural conformation is found to be vital for the formation of key H-bonds within the binding pockets of several kinases, including B-Raf, BCR-ABL, and KIT . Upon evaluation with 442 different human kinases, SHetA2(Figure 9) has exhibited good binding affinity for KIT kinase (binding constant, Kd = 820 nM). This indicates that SHetA2 is a potential candidate for kinase inhibitor development. More importantly, one of the metabolites of SHetA2 (Figure 9, 25) has also shown comparable binding affinity for KIT kinase (Kd = 1200 nM) . This suggests that other metabolites of SHetA2 may also be active, which could have acted via different mechanisms of action and contributed to the various anticancer effects observed, making it a versatile chemotherapeutic agent.
7. Metabolism of SHetA2
Using liquid chromatography and tandem mass spectroscopy, four GSH adducts were identified along with four mono- and dihydroxylated SHetA2 metabolites . At least one of these metabolites (Figure 9, 25) has been deemed active against KIT kinase as mentioned earlier. Apart from the hydroxylated metabolites, other metabolites of SHetA2 were also detected
Given that ShetA2 has several mechanisms of action involving various cellular targets, these findings suggest the possibility for SHetA2 to be metabolized into several active metabolites, each targeting a different molecular pathway. This is further supported by a recent
|1||Oral bioavailability at 20 mg/kg||15%|
|Oral bioavailability at 60 mg/kg||19%|
|2||Urinary excretion (%)||Not reported|
|3||Bound to mouse and human plasma proteins at μM concentrations||99.3–99.5%|
|4||Total body clearance (L/h/kg)||1.8|
|5||Volume distribution (L/kg) at steady state (
|6||Half-life in mouse plasma||12.7 h|
|Half-life in the mouse||Detectable after 60 h after IV administration|
|Half-life in human plasma (once a day dosing proposed)||12 h|
|7||Peak time (plasma concentrations of 10 μM following i.v. bolus dose)||5 min|
|8||Peak concentration (IV 20 mg/kg dose of SHetA2 to mice)||10 µM after 5 min|
|Maxima mean plasma concentration in the mouse (PO)||0.79 µM at 2 h|
|2.35 µM at 3 h|
9. Possible clinical applications
SHetA2 is now in Phase-0 clinical trial for ovarian cancer chemoprevention. However, since it inhibits growth of most cancers
|Ovarian cancer||G1-phase cell cycle arrest through cyclin D1 degradation and apoptosis. Reduced cell survival, and increased sensitivity to TRAIL- and TNFα-induced apoptosis. Induced glandular differentiation reversed cancerous phenotype.|||
||SHetA2 decreased survival of all three cell lines, but did not increase sensitivities of cell lines to chemotherapeutic drugs.|||
|Lung cancer (NSCLC)||Increased sensitivity to TRAIL-induced apoptosis through modulation
of c-FLIP and upregulation of DR5. Inhibits growth, triggers ER stress.
|Head and neck cancer (HNSCC)||Apoptosis in 8 HNSCC cell lines.|||
|Cervical cancer||Growth inhibition by micromolar concentrations.|||
|Kidney cancer||Induced G1 cell cycle arrest in Caki-1 and normal HK-2 cell lines; decreased cyclin D expression; induces apoptosis.|||
SHetA2 has exhibited anticancer activity in 60 NCI cancer cells and has shown favorable results in inhibiting various types of cancer growth
|c-FLIP:||Cellular FLICE-inhibitory protein|
|DR5:||Death receptor 5|
|EC50:||50% Effective concentration|
|FDA:||US Food and Drug Administration|
|GI50:||50% Growth inhibition|
|NCI:||National Cancer Institute|
|MTD:||Maximum tolerated dose|
|RAR:||Retinoic acid receptor|
|RAID:||Rapid access to intervention development|
|RAPID:||Rapid access to preventive intervention development|
|ROS:||Reactive oxygen species|
|RXR:||Retinoid X receptor|
|SAR:||Structure activity relationship|
|TRAIL:||Tumor necrosis factor-related apoptosis-inducing ligand|
|TTNPB:||(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid|
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