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1. Introduction

The immune system plays a dual role in cancer under the concept of the immune-editing theory (1-3). As such, the immune system can fight cancer, but can also shape cancer immunogenicity. In the first phase, the immune system may eliminate cancer, while in a later phase, such as in the equilibrium phase, a balance is established between antitumor and pro-tumor immunity until cancer cells escape the immune control and acquire metastatic potential (1-3). This underscores the importance of immunomodulatory therapy in boosting endogenous defense mechanisms to fight cancer and infections.

Immunomodulatory drugs include agents that modify the immune response by increasing (immunostimulators) or decreasing (immunosuppressives) the production of serum antibodies (4). Cancer-targeted small molecule-based immunomodulators are classified as inhibitors, agonists, or degraders (5). Another classification of cancer immunotherapeutics include checkpoint inhibitors, chimeric antigen receptor T-cells (CAR T-cells), monoclonal antibodies, cancer vaccines, cytokines, radio-immunotherapy and oncolytic virus therapy (6). Immunomodulators, one of four immunotherapeutic classes, are themselves divided into four categories based on their mode of action as follows: Checkpoint inhibitors, cytokines, agonists and adjuvants. To date, 16 different immune-modulators [nine checkpoint inhibitors (7-10)], four cytokines (11-13) two adjuvants (5) and a small molecule with immunomodulatory properties (14) have been approved by the US Food and Drug Administration (FDA) for the treatment of several major cancer types. Imiquimod, one of the two approved adjuvants (5), targets Toll-like receptor (TLR)-7 and activates pathways involved in the innate immune system, which results in the induction of an adaptive immune response (15,16). In addition to its topical application for anal and genital warts, and actinic keratosis (17), imiquimod is used and approved for subsets of patients with basal cell carcinoma.

Imidazoquinoxalines, imiquimod analogues, have outperformed their parent compound and have exhibited potent antitumor properties with less adverse events in the preclinical research setting (18,19). In particular, the most promising analogues, from first and second generations, EAPB0203, EAPB0503 and EABP02303, have been evaluated in different types of cancer, such as melanoma (20-22), adult T-cell leukemia/lymphoma (ATL) (19,23), chronic myeloid leukemia (CML) (24) and acute myeloid leukemia (AML) (25). In addition, they have exhibited anti-parasitic activity, specifically against cutaneous leishmaniasis (26). At the molecular level, EAPB0203 and EAPB0503 act on tubulin, inhibit its polymerization, and consequently arrest the cells in the G2/M phase (19,24,25), possibly leading to apoptosis (19,22). However, and unlike the two former compounds, EABP02303 does not bind to tubulin and its specific mode of action remains to be elucidated.

In the sections below, an overview of imiquimod is provided, including a detailed description of the chemical synthesis of imidazoquinoxalines and their therapeutic applications to date.

2. Imiquimod

Imiquimod (1-(2-methylpropyl)-1H-imidazo(4,5-c) quinolin-4-amine), also known as S-26308 or R-837, is the first non-osidic (i.e., contain no sugar) nucleoside analogue of the imidazoquinoline family. It is a tricyclic organic molecule with a nitrogen (N)-containing heterocyclic compound of four N atoms, a quinoline component, a 1H-imidazole ring, and a methyl-propyl group (27). Imiquimod belongs to the class of immune response modifiers (15,28), and is commercialized under the name of Aldara® or Zyclara®. Its topical use was approved by the US FDA in 1997, for the treatment of certain viral infections, such as perianal and genital human papilloma virus (HPV) disease (genital warts) by stimulating the host immune system (29,30). Imiquimod was also the first immune response modifier used for the treatment of infectious skin conditions due to its potent in vivo anti-viral and antitumor activities (16). In addition, it was effective as a topical therapy for certain types of skin cancer, including superficial basal cell carcinoma, Bowen's disease, superficial squamous cell carcinoma, certain superficial malignant melanomas and actinic keratosis (15,31-33). The systemic administration of imiquimod in mice has been shown to exert potent antitumor effects against various types of cancer, such as melanoma, lung sarcoma, mammary, colon, bladder carcinoma (19,34), basal cell carcinoma (16,35), actinic keratosis (36) and cutaneous B-cell lymphoma (19,33,37). Furthermore, its therapeutic spectrum has been extended to certain parasitic infections, such as cutaneous leishmaniasis (26,38) and toxoplasmosis (39). In a previous study on cutaneous leishmaniasis, imiquimod was clinically used, in combination with a systemic antimonial, and yielded a cure rate of 90% in patients with refractory cutaneous leishmaniasis, as compared to pentavalent antimonial treatment alone (40). In a clinical trial performed in Peru, Miranda-Verastegui et al (41) demonstrated that this combination was more effective than the placebo plus pentavalent antimony (41). Imiquimod has also been proven to be effective as a first line therapy for cutaneous leishmaniasis (42).

Chemical synthesis of imiquimod

Over the years, the chemical synthesis of imiquimod was performed using several methods. Conventional synthesis was based on the addition of imidazole group to the quinoline core (27) (Fig. 1).

Figure 1 Conventional method for imiquimod synthesis (27). First, 4-chloro-3-nitroquinoline undergoes a nucleophilic aromatic substitution reaction, with isobutylamine to substitute isobutylamine for chlorine (Cl). This reaction is followed by the hydrogenation of the nitro group to form the imidazole ring in the resultant diaminoquinoline scaffold treated with triethyl orthoformate in glacial acetic acid to form the tricyclic core. Finally, imiquimod is obtained by the addition of the side amino group. The latter was added through a three-step sequence of reactions which involve formation of an N-oxide intermediate, chlorination and amination.

However, this method presented several limitations, including the numerous and lengthy steps involved in the addition of N atoms. Thereafter, other strategies were adopted to save time and cost. One strategy began with 2-bromobenzaldehyde instead of quinolone (Fig. 2), yet it resulted in a low yield of 30%. Another approach used, in the first step, anthranilic acid, a commercially available compound. Several intermediates including benzoxazine, hydrazoic acid, tetrazoloquinoline and iminophosphorane were produced, and imiquimod was formed from iminophosphorane. While this route was synthetically interesting, it included the formation of a tetrazole derivative, a potentially explosive product (27).

Figure 2 Synthesis of imiquimod. First, 2-bromobenzaldehyde is coupled with ethyl isocyanoacetate via a Knoevenagel coupling reaction and the resulting acrylate is brominated, and oxidized, followed by the addition of isobutylamine. This generates the imidazole ring. Then, several reactions are used to cyclize the quinoline moeity and provide the two missing N atoms: One N is added by a series of reactions replacing the ethyl-ether group by an amide forming group then the other N and the primary amide are added after a copper-mediated N-arylation reaction and Br substitution rendering the imidazoquinolinone scaffold (27,118).

Given the limitations of the aforementioned methods, new strategies are being developed to improve imiquimod synthesis, and to generate imiquimod derivatives with enhanced efficiency and reduced adverse effects (discussed below).

Biological activity and mechanisms of action of imiquimod

The exact mechanisms of action of imiquimod have not yet been fully explored. Nevertheless, it is well documented that imiquimod is a TLR-7 agonist (15,43), which ligates TLR-7 activating immune cells. TLR-7 is expressed on the endosomal surface of antigen presenting cells and is commonly involved in pathogen recognition (40). Other cell types activated by imiquimod include natural killer cells, macrophages, dendritic cells and B-lymphocytes, leading to the consequent activation of downstream protein kinases and transcription factors, including nuclear factor-κB (NF-κB), and the production of pro-inflammatory cytokines and chemokines, including tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-8(44), IL-12, interferon (IFN)-α and IFN-γ (19,45-53). Cytokine induction and cellular infiltrates underlying cell-mediated immune responses result in the regression of warts caused by HPV infection (54). In the case of cutaneous leishmaniasis, imiquimod exhibits an anti-leishmanial activity via the activation of TLRs, the production of inflammatory cytokines and the induction of nitric oxide release (26). Recently, the mechanism of action of imiquimod against acute and chronic toxoplasmosis was elucidated. Indeed, imiquimod was shown to upregulate the expression of TLR-7, -11 and -12, and subsequently mediate the activation of the MyD88 pathway, resulting in the induction of an immune response (39). There is also evidence to indicate that imiquimod, when applied to the skin, can lead to the activation of Langerhans cells, which then migrate to local lymph nodes to activate the adaptive immune system (40).

Independent of TLR-binding, imiquimod can also boost pro-inflammatory signaling by restricting the negative feedback on inflammation. In that sense, imiquimod inhibits adenylyl cyclase activity and consequently suppresses the adenosine receptor signaling pathways involved in negative regulation of inflammation (51,55). Imiquimod has also been shown to inhibit tumor progression in immunocompromised animals (34), and its topical application potently inhibits tumor induced-angiogenesis in vivo, in a dose and time-dependent manner (56). This effect was associated with the imiquimod-induced production of several cytokines, including IFN-γ, TNF-α and IL-18. This constitutes an effective response to the treatment of HPV-associated benign and premalignant tumor, but also of early malignant angiogenesis-dependent proliferative tumors (56).

Under the TLR-independent pathway, imiquimod induces direct apoptotic effects, through the intrinsic mitochondrial-and Bcl-2-dependent apoptotic pathway (18,50,57), leading to the translocation of cytochrome c into the cytosol and the activation of procaspase-3 and -9(58). Notably, imiquimod selectively induces the apoptosis of transformed keratinocytes and melanoma cells, without any effect on their normal counterparts (19,49-51,59).

Clinical use of imiquimod and adverse effects

Imiquimod is approved and commercially available as a 2.5, 3.75 or 5% cream for the treatment of external genital warts and superficial basal cell carcinoma. However, it is also used for off-label conditions as follows: Melanocytic proliferations, such as lentigo maligna (60-62), atypical nevi (63), as well as lichen sclerosus, alopecia areata (64), Kaposi sarcoma (65,66) and Molluscum contagiosum (67). Research on imiquimod is still an active area. Indeed, >450 studies were published on the topic over the past year alone (as of May, 2023). A previous study evaluated the potentiating effect of imiquimod on dyphencyprone for the topical immunotherapy of alopecia areata and reported that 77% of patients exhibited an improvement in symptoms upon the addition of imiquimod (68). Imiquimod was also tested as a topical treatment in patients with HIV and high-grade squamous intraepithelial lesions, susceptible to develop into anal cancer (69). Compared to the active monitoring group, the rate of anal cancer onset was 57% lower in the treatment group; suggesting that the treatment of high-grade squamous intraepithelial lesions with medications such as imiquimod is protective against anal cancer (69). Another recent study described the efficacy of imiquimod loaded in phospholipid-free small unilamellar vesicles targeted to hepatocytes, in the treatment of chronic hepatitis B (70). In patients with lentigo maligna, imiquimod was shown to reduce the lentigo maligna surface after 1 month of treatment and could be prescribed to prevent the progression of these lesions to carcinogenesis (71). In a human melanoma cell line, imiquimod induced cell death by lysosomal membrane permeabilization and the release of lysosomal proteolytic enzymes, including cathepsins, which resulted in mitochondrial dysfunction (72). While imiquimod is an effective and safe therapy, certain adverse effects have been reported; these include local inflammatory reactions such as itching, burning, bleeding, erosions, ulcerations, excoriations, crusting, induration, edema and pain (73,74). Some patients have presented with systemic reactions, such as upper respiratory tract infections and flu-like symptoms, such as fever, sinusitis, headaches and tiredness (50,74,75). In the case of periocular skin lesions, imiquimod may cause conjunctivitis and ocular stinging (61). In addition, imiquimod can affect vitamin B12 levels (60). Imiquimod has also been shown to be associated with hypertrophic lupus erythematosus in an elderly patient (76). Additionally, a case report of actinic keratosis associated imiquimod with a severe case of bullous pemphigoid (77) and the occurrence of lupus-like reactions (78). In another study, when applied to a to a single lesion of in situ melanoma in a patient, imiquimod caused debilitating severe fatigue (79). Moreover, imiquimod caused localized skin ulceration in a patient with type-2 diabetes mellitus (80). Finally, women of reproductive age are advised to use contraception upon imiquimod treatment, due to the absence of definitive data on its teratogenic potential (74).

3. Quinolines and quinoxalines

Heterocyclic compounds are a cornerstone of current anticancer drug design due to their favorable pharmacokinetic and pharmacodynamic properties (higher lipophilicity, adjusted polarity and other physicochemical features including solubility, ionization state and hydrogen-bonding capacity). In 2015, ~30% of US FDA-approved anticancer drugs included one or more cyclic rings containing a nitrogen or an oxygen (81).

Quinolines and quinoxalines are N-containing heterocycles found in a number of natural compounds. In both quinoline and quinoxaline compounds, the main core of imiquimod is composed of a benzene ring. The difference is that, in quinoxalines, the pyridine ring of quinoline is replaced by a less alkaline component, the pyrazine ring (Table I) (82-84).

The quinoline compound has limited applications, while its derivatives span a wide range of activities, such as anti-malarial (quinine, quinidine, chloroquine, mefloquine, amodiaquinine, primaquine, etc.), anti-viral (saquinavir), anti-bacterial (fluoroquinolones such as ciprofloxacin, sparfoxacin, gatifloxacin, etc.), anti-fungal/anti-protozoan (clioquinol), anti-helminthic (oxamniquine), local anesthetic (dibucaine), anti-asthmatic (montelukast), anticancerous (camptothecin, irinotecan, topotecan, etc.), anti-psychotic (aripiprazole, brexpiprazole, etc.), anti-glaucoma (carteolol) and cardiotonic (vesnarinone) activities. In the particular case of cancer, quinoline derivatives induce cell cycle arrest and apoptosis, the inhibition of angiogenesis, the disruption of cell migration and the modulation of nuclear receptor responsiveness in cancer cells (85). Similarly, quinoxaline compounds exibit a wide array of pharmacological activities, some of which include anti-malarial (86), anti-inflammatory (87), anti-HIV (88) and anticancer activities (89).

4. Imidazoquinoxalines

Imiquimod exerts a selective cytotoxic effect on cancer cells. However, the imiquimod-induced production of pro-inflammatory cytokines can have deleterious effects. Hence, a series of imiquimod analogues belonging to the quinoxaline family was synthesized, with the ultimate aim of enhancing the antitumor potential of imiquimod, while rendering it systemically available and curbing its toxicity profile (18).

A series of three imiquimod analogues were synthesized: The imidazo[1,2-a]quinoxalines, the imidazo[1,5-a]quinoxalines and the pyrazolo[1,5-a]quinoxalines. The present review focuses on three compounds of the imidazo[1,2-a]quinoxalines series, namely EAPB0503, EAPB0203 and EAPB02303. The first generation encompasses EAPB0203 [N-methyl-1-(2-phenylethyl) imidazo[1,2-a]quinoxalin-4-amine)] as an earlier derivative, followed by EAPB0503 [1-(3-methoxyphenyl)-N-methyl imidazo[1,2-a]quinoxalin-4-amine] (Fig. 3), and it paved the way for in vitro studies revealing considerable anticancer potential for this class of imidazoquinoxalines (19).

Figure 3 Structure of imiquimod, EAPB0203, EAPB0503 and EAPB02303. The heterocyclic scaffold platform of imiquimod is comprised of a quinoline component (a six-membered ring, lowermost ellipse) and a 1H-imidazole ring (a five-membered ring, middle ellipse). Analogue design is based on a modified heterocyclic scaffold, named imidazoquinoxaline, comprised of a quinoxaline (bicycle with two nitrogen atoms) fused on one of its face to an imidazole, leading to the presence of a bridgehead nitrogen which slightly modifies the general planarity of the molecule (90). The isopropyl group in imiquimod (uppermost ellipse) is substituted by a phenethyl in EAPB0203, a 3-methoxyphenyl in EAPB0503 or a 3,4-dihydroxyphenyl group in the new derivative EAPB02303. In the three imidazoquinoxaline analogues, the amino group of imiquimod is replaced by a methylamino group.

Recently, a newer imidazoquinoxaline derivative, EAPB02303 [1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxaline] (Fig. 3, right panel), as the lead compound of the second generation, was synthesized; this demonstrated a higher potency in a melanoma cell line model, with a 50% inhibitory concentration (IC50) of 3 nM, as compared with IC50 of 383 nM for EAPB0503(90).

Chemical synthesis of EAPB0203, EABP0503 and EAPB02303

EAPB0203 was first synthesized using the traditional method, which consists of the condensation of two imidazole derivatives coupled to ortho-fluoroaniline, followed by an intramolecular cyclisation and substitution with the appropriate amines (19). The synthesis of EAPB0503 is based on EAPB0203 synthesis, but was modified and optimized using the Suzuki reaction under basic conditions in a microwave-assisted reaction to yield greater purity with shorter synthesis time (Fig. 4). In summary, EAPB0503 was generated by replacing the phenethyl group in EAPB0203 with 3-methoxyphenyl (Fig. 3) (22). As regards EAPB02303, it was obtained by the substitution of 3-methoxyphenyl group at R1 position in EAPB0503 by 3,4-dihydroxyphenyl group on the 4-methylaminoimidazo(1,2-a)quinoxaline heterocyclic platform (Fig. 3) (90).

Figure 4 EAPB0503/EAPB02303 synthesis steps. Carbonyl-imidazole dimer that results from the bimolecular condensation of 2-imidazole carboxylic acid is coupled with ortho-fluoroaniline to give an intermediate, which is then cyclized to afford the new tricyclic core. Further treatments yield N-methylimidazo(1,2-a)quinoxaline-4-amine and Suzuki coupling performed on the 1-bromine derivative leads to either EAPB0503 or EAPB02303 products.

Pharmacokinetic and pharmacodynamic properties of EAPB0203, EAPB0503 and EAPB02303

The modifications introduced into the imidazoquinoxalines, EAPB0203, EAPB0503 and EAPB02303(19), aimed to improve their polarity and binding properties.

Imidazo[1,2-a]quinoxalines, mostly EAPB0203 and EAPB0503, their metabolites, and their major oxygenated derivatives, were characterized and identified using nuclear magnetic resonance and liquid chromatography-mass spectrometry studies (91). Briefly, EAPB0203 and EAPB0503 are metabolized inside liver microsomes of numerous species, including mouse, human, dog and rat. Both EAPB0203 and EAPB0503 metabolites exhibit an increased polarity compared to their parent compounds, and have mostly shorter half-lives, apart from one metabolite (92). EAPB0203 and EAPB0503 biotransformation began with a demethylation step, yielding their two main metabolites EAPB0202 and EAPB0603, respectively. These metabolites are less potent than their parent molecule (92). Furthermore, ~98% of EAPB0203 and EAPB0503 bind to human serum albumin in the plasma, with very little compound remaining in the free form. Both compounds are eliminated from the plasma in two phases: an initial rapid decline phase, followed by a prolonged terminal phase. The terminal half-life is ~2 h. However, the systemic availability of EAPB0203 and EAPB0503, upon intraperitoneal administration is relatively modest, at 22.7 and 35% respectively (92); this motivates further research to enhance bioavailability and pave the way for more preclinical (and ultimately clinical) studies.

5. Immunomodulatory effect

A total of 22 imidazo(I,2-a)quinoxalines derivatives have been proven to be potent inhibitors of nucleotide phosphodiesterase enzyme 4 extracted from human alveolar epithelial cells (93). It is considered that by inhibiting phosphodiesterase enzyme 4 activity, these compounds could lead to cAMP accumulation and transcription factor cAMP response element-binding protein activation inside the cell (31). In addition, since they are able to decrease TNF-α levels (19,93) they can consequently, modulate cytokine synthesis and/or production by immune cells, exerting therefore their anti-inflammatory activity (93). Moreover, it has been shown that imidazo[1,2-a]quinoxalines activate the p38 MAPK pathway and inhibit the PI3K pathway in a murine fibroblastic cell line (93).

6. Antitumor properties of EAPB0203, EAPB0503 and EAPB02303

Melanoma

Melanoma is the most aggressive form of skin cancer and is associated with a poor prognosis (94). Standard of care begins with surgical excision (when the tumor is resectable), systemic treatment (chemotherapy and/or immunotherapy), or radiation therapy for unresectable tumors or as adjuvant therapy (21). However, the mortality rates remain very high, urging the need for the identification of novel therapeutic targets. The activity of imiquimod and some of its derivatives was previously tested in melanoma cells in vitro and in animal models in vivo (50,95). EAPB0203 was 50-fold more potent than imiquimod and exhibited a remarkable cytotoxic activity against A375 melanoma cell line (19,96). EAPB0203 was also 110-fold more potent than fotemustine, a major treatment for metastatic melanoma (19,97). Of note, as previously demonstrated EAPB0503 exhibited a 7-9-fold greater antitumor and cytotoxic activity than EAPB0203 in the same cell line, and induced apoptosis by activating the NF-κB pathway (92). Indeed, both EAPB0203 and EAPB0503 induced the intrinsic apoptotic pathway characterized by the loss of mitochondrial membrane potential, the release of cytochrome c, the activation of caspases and the subsequent fragmentation of DNA. The potent cytotoxic activity of EAPB0503 was also observed in an in vivo mouse model of xenograft melanoma (19,96). In this regard, the antitumor activity of APB0503 was attributed to anti-proliferative and anti-mitotic effects, mediated by the inhibition of tubulin polymerization (18,22). This compound interacts with tubulin via a colchicine-binding site (18), with a 52% higher potency than colchicine, a well-known natural inhibitor of tubulin polymerization. Unlike imiquimod, the effect of EAPB0503 on tubulin polymerization in melanoma cells is not associated with its TLR-7 agonist activity, even at concentrations >300 µM (18). An EAPB0503 derivative, 1-(2-hydroxy-3-methoxyphenyl)-N-methylimidazo [1,2-a]quinoxalin-4-amine, exhibited a greater inhibition of tubulin polymerization in melanoma A375 cells (22).

Given encouraging data on melanoma in vitro and in vivo models, first-generation imidazoquinoxalines were evaluated in models of hematological malignancies (25) .

The next-generation derivative, EAPB02303, was unable to bind to tubulin at the colchicine site (90). Notably, EAPB02303 was still able to inhibit the growth of A375 melanoma cells in vitro at a nanomolar concentration range, independent of the inhibition of tubulin polymerization. The growth inhibition in vitro was, dose-dependently, associated with a reduction in size and weight of mouse-xenograft A375 melanoma cell tumors. In addition, growth inhibition was associated with a lower mitotic index, but not with necrosis (90). Transcriptomic analysis, in comparison with first-generation imidazoquinoxalines and a panel of 12 well-known anticancer drugs, suggested that EAPB02303 acts through a different mechanism. However, the exact mechanistic pathway remains to be elucidated (90).

Of note, the second-generation derivatives, EAPB02302 and EAPB02303, were even more cytotoxic on A375 cells, with IC50 values of 60 and 10 nM, respectively, versus an IC50 value of 1,570 and 200 nM for EAPB0203 and EAPB0503, respectively (96).

ATL

ATL is a rare and aggressive blood malignancy due to the transformation of T-cells by human lymphotropic virus type I. ATL is associated with a poor prognosis, due to chemo-resistance and immunosuppression (98). Following more than four decades of research, the therapeutic management of ATL remains intricate and a cure for ATL is still out of reach in the majority of patients. The current strategies include the watch-and-wait policy, conventional chemotherapy, allogeneic hematopoietic cell transplantation (allo-HCT), and a combination of two antiviral agents, zidovudine and IFN-α (99-103). Although indolent ATL and a fraction of acute ATL exhibit a long-term survival subsequent to antiviral therapies and one third of patients with aggressive ATL undergo allo-HCT and <10% of those who received chemotherapy could have disease control for >5 years.

Several targeted therapies have been tested in ATL. Among these, EAPB0203 has been shown to induce growth inhibition, cell cycle arrest and apoptosis, selectively in ATL cell lines, while no effect has been reported in normal resting or PHA-activated peripheral blood mononuclear cells taken from two healthy donors (23). EAPB0203 induces apoptosis by decreasing the levels of the anti-apoptotic proteins, c-IAP-1 and Bcl-xL, thus triggering the intrinsic apoptotic pathway. In addition, EAPB0203 treatment stabilizes the tumor suppressor proteins p21 and p53 in a dose-dependent manner (19).

CML

CML is a clonal myeloproliferative disorder resulting from a reciprocal translocation between the abl proto-oncogene on chromosome 9 and the breakpoint cluster region (bcr) on chromosome 22, resulting in a shortened chromosome 22, known as the Philadelphia (Ph) chromosome. This translocation produces the recombined bcr-abl gene, encoding for BCR-ABL, a constitutively activated tyrosine kinase, responsible for CML leukemogenesis, maintenance and progression of the disease from chronic to blast phase, indicating a poor prognosis. The initial treatments for CML included busulfan and hydroxyurea. These were later replaced with IFN-α (104). In 2001, CML treatment witnessed a revolution with the use of imatinib mesylate (imatinib), the first tyrosine kinase inhibitor (TKI) (105-107). However, this treatment was associated with certain drawbacks, including intolerance to treatment, the lack of a therapeutic response or resistance, and this has motivated the development of second- and third-generation TKIs.

While TKIs significantly revolutionized CML treatment and prolonged survival of patients, CML cure is still out of reach, and bone marrow transplantation remains the only long-term curative option for CML patients. Both EAPB0203 and EAPB0503 were evaluated in an in vitro model of human blast crisis CML cell lines. The compounds induced cell cycle arrest at the G2/M phase, accompanied by an increased level of histone 3 phosphorylation. Growth inhibition was more pronounced in three chronic-phase CML cell lines treated with EAPB0503 as compared to EAPB0203(24), and was enhanced by the induction of the intrinsic apoptosis pathway. Molecular analysis revealed that EAPB0503-treated CML cells exhibited decreased BCR-ABL oncoprotein levels, which could explain growth inhibition. Notably, EAPB0503 appeared to circumvent imatinib resistance in vitro, presenting it as a potentially attractive therapeutic option in CML. The combination of imatinib (CML standard-of-care) and EAPB0503 synergized to inhibit CML cell line growth and proliferation in vitro (24). Collectively, these findings suggest that EAPB0503 is a potential therapeutic agent capable of inhibiting CML cell growth, synergizing with imatinib to alleviate the tumor burden, and overcoming resistance to imatinib and maybe other TKIs.

AML

AML is a complex and heterogeneous hematological malignancy, characterized by the excessive proliferation of undifferentiated myeloid precursors, resulting in an impaired hematopoiesis and bone marrow failure. AML is associated with a highly variable, yet frequently poor prognosis, based on somatic genetic alterations (108). Due to the genetic complexity and heterogeneity of AML, treatment strategies remain variable among patients, apart from the AML subtype, acute promyelocytic leukemia and core binding-factor (CBF) AML, which have their own standard of treatment (109,110). In general, AML treatment remained unaltered for more than three decades, and relied on aggressive chemotherapy administered in two phases: An induction phase to eliminate AML blasts, and a consolidation phase to prevent relapse (111). Hematopoietic stem cell transplantation was used independently or sequentially, depending on the availability of stem cell donors and the overall clinical fitness of the patient (112).

Genome-wide studies, including gene and microRNA expression profiles, single nucleotide polymorphisms and gene copy number studies, have identified a growing number of recurrent genetic mutations of AML (113). Among the identified genes, mutations of nucleophosmin-1 (NPM-1) and fms-like tyrosine kinase 3 (FLT3) are prognostic markers of AML. NPM-1 is one of the most frequently mutated genes in AML (114). NPM-1 mutations alter the C-terminal DNA-binding domain of the protein, resulting in an aberrant nuclear export sequence and its retention in the cytoplasm, hence the NPM-1c nomenclature, referring to its cytosolic localization (115).

The pre-clinical efficacy of EAPB0503 and EAPB0203 has been evaluated in AML. EAPB0503 was shown to significantly and selectively inhibit the growth of NPM-1c-positive cells, at lower concentrations than EAPB0203(25). Growth inhibition was associated with EAPB0503-induced early apoptosis in NPM-1c-, but not wild-type NPM-1-AML cells through caspase activation and p53 activation (25). More recently, EAPB0503 was shown to prolong the survival of mice with NPM-1c AML xenografts and a novel mechanism of action of this compound was found. Indeed, EAPB0503 induced NPM-1c SUMOylation, concomitant with the downregulation of the de-SUMOylase, sentrin/SUMO specific peptidase 3, and paralleled by the upregulation of alternative reading frame (ARF) in NPM-1c-expressing cells (116). These results implicate EAPB0503 in the post-translational modifications of NPM-1c, demonstrating that therapies modulating NPM-1c post-translational modifications can be introduced to the management of NPM1c AML.

In conclusion, EAPB0503 displays a greater anti-leukemic activity than EAPB0203 in both CML and AML. This is hypothesized to be due to the ethyl linker that separates the imidazoquinoxaline heterocycle from the phenyl group in EAPB0203, but not in EAPB0503(24).

7. Anti-parasitic activity of EAPB0503

The anti-parasitic activity of imidazoquinoxalines, was first reported for imiquimod (26). In the context of cutaneous leishmaniasis, imiquimod exerted its anti-amastigote activity via TLR-7 upregulation, leading to NF-κB activation and pro-inflammatory cytokine production. However, the effect of EAPB0503 on TLR-7 was less prominent (26). Noteworthy, findings suggest that EAPB0503 may act not only via TLR-7, but also through other TLRs (26). Moreover, the levels of macrophage inflammatory protein-1α and β, monocyte chemoattractant protein and some pro-inflammatory cytokines, such as IL-1β, IL-1, IL-6, IL-12 and TNF-α, which appear to be associated with resistance against leishmaniasis, are increased upon exposure to EAPB0503. This increase appears to be crucial for the clearance of cutaneous leishmaniasis and for the protective role of imiquimod derivatives against cutaneous leishmaniasis (26). Moreover, the production of the inducible nitric oxide synthase, normally induced in response to pro-inflammatory cytokines, increased in response to EAPB0503 further enhancing the leishmanicidal activity of macrophages infected with Leishmania spp. By contrast, anti-inflammatory cytokine levels, such as IL-10 and IL-4, are usually associated with disease progression (117), decreased by 4- and 15-fold, respectively, upon EAPB0503 exposure versus 4-fold upon exposure to imiquimod in L. tropica-infected macrophages (26). Moreover, EAPB0503 exhibited a more prominent anti-leishmanicidal activity than the clinically used glucantime, whether alone or combined with imiquimod (26)

8. Conclusions and future perspectives

As an immune response modifier, imiquimod has shed light on a new class of potential anti-cancer agents. Imidazoquinoxalines, imiquimod analogues, were synthesized in an attempt to mitigate the deleterious effects of the imiquimod-induced production of pro-inflammatory cytokines and to improve its antitumor properties. Earlier agents, EAPB0203 and EAPB0503, exhibited notable antitumor properties, mediated by the inhibition of tubulin polymerization, the inhibition of growth and the induction of apoptosis, in melanoma and/or leukemia models. In addition, they exhibited a potential to synergize with standard-of-care treatments. The next-generation derivative, EAPB02303, exhibited a more potent cytotoxic activity against melanoma cells, through a mechanism, which is independent of tubulin polymerization and which remains to be elucidated. A summary of the mechanisms of action of imiquimod and its analogues/derivatives is provided in Table II. Currently, imidazoquinoxalines are actively assessed in preclinical models of diseases, mainly cancer, in order to provide sufficient scientific evidence and promote their translation from bench to bedside.

Table II Mechanisms of action of imiquimod, EAPB0203, EAPB0503 and EAPB02303.

Table II

Mechanisms of action of imiquimod, EAPB0203, EAPB0503 and EAPB02303.

Compound	Disease/disease model	Mechanisms of action	(Refs.)
Imiquimoda	Genital warts	TLR-7: Innate and adaptive immune response activation	(15,43)
	Cutaneous	and cytokines secretion	(38)
	Leishmaniasis	Apoptosis: Bcl-2-dependent intrinsic apoptotic pathway	(26)
	Toxoplamosis	Nitric oxide production in response to pro-inflammatory cytokine	(39)
EAPB0203	ATL	Growth inhibition: Inhibition of tubulin polymerization	(19)
	Melanoma	Apoptosis: Anti-apoptotic proteins c-IAP-1 and Bcl-xL, downregulation triggering the intrinsic apoptotic pathway	(19)
EAPB0503	AML	Growth inhibition: Inhibition of tubulin polymerization	(25)
	CML	Apoptosis: Intrinsic apoptotic pathway	(24)
	Melanoma		(22)
EAPB02303	Melanoma	Growth inhibition: Unknown mechanism; independent from tubulin binding	(90)

[i] ^aCurrently used in clinical practice. TLR-7, Toll-like receptor 7; AML, acute myeloid leukemia; ATL, adult T-cell leukemia/lymphoma; CML, chronic myeloid leukemia.

Acknowledgements

The authors would like to acknowledge Miss Christa-Maria El-Khoury, Miss Nesrine Dagher and Miss Reem Hamze for contributing to the literature review, which was in partial fulfilment of their graduate research projects, at the Department of Biology, Faculty of Sciences, Lebanese University, Beirut, Lebanon.

Funding

Funding: No funding was received.

Availability of data and materials

Not applicable.

Authors' contributions

JO wrote the manuscript. Authors AS, HEH, CDM, PAB, MES and JS reviewed the different drafts, contributed to the writing and synthesized the available literature. JS designed the review and oversaw the writing process. All authors have read and approved the final manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References