Unlocking the power of imidazoquinolines: recent advances in anticancer and immunotherapeutic strategies

ABSTRACT

The challenges in drug discovery aiming to mitigate cancer progression are the thrust area of scientific research for several decades. Since the advent of heterocyclic chemistry, drug discovery programs have made significant achievements that lead to the development of numerous drugs with broad spectrum of potencies, contributing to both diagnostic and therapeutic advancements. Till date, efforts to discover more potent and efficient drug candidates are underway to minimize adverse side effects of existing chemotherapeutics. In view of the above, small-molecule agonists that can interact with different immune modulators like toll like receptor-7 (TLR-7) and TLR-8 are being investigated and explored. These candidates are expected to display profound effect on anti-tumoral activity by enhancing the production of proinflammatory cytokines. Recently, numerous imidazoquinoline derivatives with proven TLR agonist activities have emerged as promising anticancer therapeutics. With advancements in technology and the evolution of new scopes in drug discovery, different strategies are being adopted, particularly with the help of nanotechnology, immune-technology, combination drug chemistry, etc., to curb the progression of various types of cancers. Herein, the novel strategies for cancer therapeutics with imidazoquinolines reported in the last 5 years, their structure-activity relationship along with important synthetic schemes for important TLR agonists, are discussed.

Graphical abstract

1. Introduction

Till date cancer is considered as the second most deadly and life threatening disease. The elusive nature of cancer and its propagation still remain inexplicable in spite of the development of numerous theories and advancement of technologies [1]. The mortality caused by cancer has always been increasing. With increasing sophistications in human lifestyle, cancer is recognized as a serious issue that has imposed severe impact on human health. Until now, there exist major challenges toward the accessible diagnostics and efficient therapeutics. Prostate, colon, breast, lung and thyroid are some of the most prevalent cancer types and particularly in children, blood cancer and cancers in brain and lymph nodes were being reported [2]. Breast cancer proliferation is considered as another serious issue that majorly affects women.

The disruption and dysfunction observed in vital genes that affect the cellular functions through induced mutations in genes is known as cancer [3,4]. Cancers alter the cell cycle that leads to abnormal proliferation. Chemical carcinogens, viruses, bacteria and radiations are some of the factors that are known to induce carcinogenesis in humans. Normal cell division and growth are assisted by proto-oncogenes which upon genetic mutation get converted to oncogenes. These oncogenes in association with tumor suppressor genes further trigger uncontrolled cell division [5].

In recent years, targeted therapeutic drugs have shown high efficacy and safety for cancer treatments as compared to traditional chemotherapy drugs [6–8]. Despite these advantages, the poor response rate and exhibited drug resistance pose major challenges toward the application of these small molecular drugs [9–11]. Zhong et al have recently reviewed the existing challenges, understanding and perspectives toward the advancement of anti-cancer drugs discovery programs [12–15]. Over the past two decades, cancer therapies have improved significantly with the replacement of broad-spectrum cytotoxic drugs with targeted treatments that specifically target cancer cells with high potency and low toxicity, while sparing normal cells [16–18]. These targeted molecular drugs are majorly categorized as small molecules and macromolecules (e.g., nucleic acids, immuno-reagents, antibody conjugated drugs, etc) [19]. There are several advantages of these small-molecule targeted drugs that include satisfactory pharmacokinetics, ease of compliance, convenient storage and transportation, etc. [20–24]. A wide range of targets were identified for these drugs that encompass various kinases, regulatory proteins, enzymes and proteasomes [25]. These small-molecule drugs are simpler in their design and primarily target proteins particularly enzymes or receptors, while having limited inhibitory effects on membrane and other secreted proteins.

Till date the demand on the progression of new strategies with precise cancer therapeutics exists [26–28]. With recent advancement in nanotechnology, efforts to address the existing limitations in therapeutic approaches were overcome with different nanoparticle-based therapies [29–31]. These nanoparticle-based drug delivery systems for cancer therapies have demonstrated excellent pharmacokinetics, precise targeting along with their ability to minimize side effects and drug resistance [32]. Most of the recently reported drug delivery systems included nano-therapeutic drugs that have found their applications in several combination therapies [31,33–36]. Examples of nanoparticles used in advanced drug delivery systems include dendrimers, quantum dots, carbon tubes, gold nanoparticles, liposome, etc.

On the other hand heterocyclic chemistry and the molecules derived from it have laid the foundation for numerous drug molecules that play significant roles in novel drug discovery programs. Particularly, literature reports are available on heterocycles containing indazoles, thiazoles, oxazoles, imidazoles, quinolines, etc. [37–40]. Based on this significance and the existing gap to overcome the limitations in drug discovery and therapeutic applications [41], fused ring systems containing quinoline and imidazole rings were considered and the recent developments made toward their applications in drug discovery are reviewed herein.

Aligning with this, heterocycles with bridgehead nitrogen were being explored extensively for their potent biological activities [42]. Bicyclic systems with heteroatoms as in imidazopyridine, pyridobenzothiadiazines, pyridobenzoxadiazines and pyridopyrimidine were reported as biologically active scaffolds for numerous applications [43]. Similarly, imidazoquinolines were also successfully demonstrated for their potential as anticancer [44], antimicrobial [45], anti-inflammatory [46], neuroprotective agents [47], etc. As part of our ongoing program for the synthesis of heterocyclic compounds and evaluation of their antitumor activities [48–50], the role of imidazoquinolines toward the therapeutic applications of cancer is reviewed from the perspective of molecular level, in order to get closer insights into the disease progression. Also the potential of recently reported imidazoquinoline derived anticancer agents against different targets and their structure activity relationships are also discussed.

2. Imidazoquinolines as inhibitors for cancer targets

Imidazoquinolines are well established immunomodulatory drugs that can effectively bind to specific receptors in the cancer pathway by mimicking their own ligands. Activation of these receptors further influences other signaling pathways to enhance antigen presentation and the activation of antigen specific T-cells [51]. Imidazoquinolines also enhance the infiltration of intra-tumoral T-cell lymphocytes further to produce proinflammatory cytokines along with the inhibition of tumor growth via induced apoptosis [52].

3. Cancer targets of imidazoquinolines

3.1. HER2

The transmembrane kinase receptor protein HER2 is one of the human EGFR families of proteins. It plays significant roles as cancer biomarker as well as target for the development of numerous diagnostics and therapeutics. Compared to normal cells, HER2 is over-expressed in human carcinomas like breast, ovarian, bladder, pancreatic, stomach and esophageal cancers. It promotes growth of cancer cells at faster pace and facilitates their spread to various parts of the body. Until now the expression levels of HER2 is monitored for prognosis. Though numerous anti-HER2 molecules were developed in the past two decades, there still exist the challenge to significantly improve the efficacies of these molecules to treat patients in their early stage of diagnosis [53–57]. Recently, several novel and advanced strategies were established to specifically target HER2 [56]. Investigations have revealed nucleic acid vaccines as more effective therapeutic toward inducing immune responses and further to resist spontaneous development of tumors [58]. Later the prospects of inducing HER2-specific immunity through nucleic acid vaccines were also revealed [59]. Further the incorporation of molecules that can influence the immune system through their regulatory and signaling roles to release immune-modulatory cytokines were known to increase the effectiveness of these vaccines. The use of these molecules as adjuvants in conjunction with nucleic acid vaccines was known to enhance the immune responses elicited by nucleic acid immunization further to stimulate antigen-specific humoral and/or cell-mediated immune responses.

Imidazoquinolines are one such immune response modifiers that were demonstrated to exert excellent anti-tumoral effects. The immune-modulating effects of imidazoquinolines are attributed to their ability to stimulate the production of endogenous cytokines, which in turn triggers innate immune responses as well as adaptive immunity in cells. The immune-modulating effects of imidazoquinolines are partially attributed to their ability to bind and activate receptors including TLR-7 and TLR-8. Numerous imidazoquinoline derivatives with proven TLR agonist activities have emerged as promising anticancer therapeutics. Some of the known side effects of TLR agonists include excessive production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IFN-γ), risk of systemic inflammatory response syndrome (SIRS), multi-organ dysfunction, leucocytosis, high risk of cardiovascular collapse, elevated liver enzymes (ALT, AST), hepatotoxicity, etc. In order to minimize these adverse effects, new methods of administering TLR agonists to enhance the specificity of these drugs toward tumor cells were researched. Moreover, the currently available or reported TLR agonists reveal several challenges including their poor pharmacokinetic stability, bioavailability along with rapid clearance from circulation that hampers their therapeutic effect. These drugs were also reported to exhibit resistance against tumor microenvironment. To overcome these limitations, new combination therapies that include the use of TLR agonists along with other methods like sonodynamic and photothermal therapy have attracted the interests of many researchers [60]. These findings highlight the potential of these molecules and related compounds to serve as adjuvants in a wide variety of vaccination strategies [61]. A recent study has demonstrated the efficacy of imidazoquinoline and its derivatives – imiquimod, gardiquimod and resiquimod to induce immune responses toward immunization [62]. (Figure 1(a))

Figure 1.

(a) Structure of important imidazoquinoline derivatives as TLR agonists. (b) Structure of imidazo[4,5-c]quinoline agonists with demonstrated selectivity toward TLR-7 receptors. (c) A series of imidazoquinoline TLR-7 agonists. (d) Variously substituted quinoline ring displaying potent anti-proliferative activity. (e) structural features in heterocyclic scaffolds displaying TLR-7/8 agonist activity [83]. (f) Structure of valine-citruline p-aminobenzyl carbamate linked imidazoquinolines.

3.2. TLR-7/8

It is known that the immune suppressive microenvironments get stimulated inside tumor cells via tumor associated macrophages and hence were considered for the development of cancer immuno-therapeutics. Small molecular TLR-7 agonists were used in cancer immunotherapy for their potential to induce the TLR-7 pathway and further to activate NF-κB and release the cytokines. But these molecules were reported with limitations due to their exerted risk of toxicity. With the development of antibody drug conjugate strategies for drug-delivery, it was possible to control the delivery of these payloads directly into the tumor cells. Cheng et al have recently reported the synthesis of a series of imidazoquinoline TLR-7 agonists via amine coupling at the benzylic moiety (Figure 1(c)) [63,64]. Further to assess the employability of these molecules for ADC applications, cell activation assays and cell permeability assays were followed.

A significant enhancement of payload efficiency was revealed upon the inclusion of hydrophobic moieties in the benzyl group further to display enhanced potency than the parent molecule (Figure 2(a,b)) [63]. Though the potency was proven with specific payloads, limitations were observed with their reactivity that projects them to be incompatible with conventional ADC linkers. This limitation was overcome by the development of new payloads following reductive amination wherein a secondary amine was utilized to alter the functional groups further to facilitate the attachment of linkers. In previous reported methods, poor nucleophilicity was observed when the amine group was attached to quinoline. The assessment of the aforementioned next-generation payloads were carried out by their ability to activate TLR-7 and induce NF-κB (Figure 2(a,b)). With this method it was possible to optimize payloads effectively.

Figure 2.

Proposed mechanism for payload release – (a) Secretion of alkaline phosphatase reporter followed by the activation of the NF-κB signaling pathway (b) Human peripheral blood mononuclear cells releasing IFNα. (c) Targeting strategy for enzyme-directed immunostimulants via covalently linked immune-stimulants as pro-immuno-stimulants. (d) Covalently linked imiquimod-galatopyranoside pro-immuno-stimulant (11) that gets converted to an active immune-stimulant in the presence of ß-galactosidase. (e) Schematic representation of directed enzyme prodrug therapy depicting the enzyme directed conversion of pro-immunostimulant into a cytotoxic payload with off-target toxicity for cells in the proximity. (f) Immune response induced via bystander effect – pro-immuno-stimulant is converted to immune-stimulant in the presence of ß-galactosidase enzyme and its diffusion activates the bystander immune cells to generate localized immune response.

3.3. Opioid growth factor receptor (OGFr)

The mechanism by which low-molecular-weight immune response modifiers, such as imidazoquinoline compounds, display potent anti-tumor properties was the focal point of research for several decades [65]. Until then research had primarily focussed on the immunomodulatory properties of this class of drugs. Later investigations have revealed that imidazoquinolines upregulate the OGFr and stimulate the interaction of OGF-OGFr axis [66–68]. The cell proliferation in this pathway is regulated by the modulating cyclin-dependent kinase inhibitors, which affect the cell cycle by halting cells at the interface of G(1)-S.

The influence of imidazoquinoline on the OGF-OGFr system has called for further investigation. It was suggested that combining OGF with imiquimod therapy could potentially enhance the effectiveness of these compounds. Moreover, the enhancement of imidazoquinoline action through OGF is expected to facilitate the reduction of imiquimod dosage and is prone to numerous side effects. Understanding the precise mechanism of action of imidazoquinoline derivatives could lead to the identification of associated compounds that can augment the growth-inhibitory activity of OGF-OGFr system without influencing pro-inflammatory effects.

3.4. PI3K/AKT/mTOR pathway

The cellular survival and proliferation is greatly influenced by the abnormal functioning of PI3K pathway that drives the progression of cancer. Therefore most of the recent drug discovery programs have focussed on this pathway as an important target for the development of therapeutics. Recently Dubey et al have investigated along this landscape of anti-cancer drug advancements, by understanding the structure-activity relationships specifically targeting the PI3K related breast cancer therapies [69]. Specific moieties, like triazines, pyrimidine, quinazoline, quinoline and pyridoxine, have exhibited promising potential as PI3K inhibitors in the combat against breast cancer. Numerous small molecule heterocyclics with proven class I PI3K inhibition potential are in the phase of clinical trials. Though these drugs are used as therapeutic agents for breast cancer they not only display various side effects but are also expensive. As a result, significant recent advancements were made toward the development of anticancer medications that aim to target the over-expression of PI3K in breast cancer.

The structure-activity relationship studies have revealed the significant role of quinoline ring for anti-proliferative activity (Figure 1(d)). When the 6^th position of quinoline is substituted with bromine, a moderate anticancer activity was observed against various cancer cell lines including A549 cells, HepG2 and PC3 (Table 1). Also when the 4^th position of quinoline ring is substituted with amines the anti-proliferative activity was significantly enhanced. It was also observed that the presence of a tertiary butyl substituted phenyl sulfonyl urea at the 3^rd position of quinoline, along with a methoxy substitution at the 6^th position, significantly enhanced the anti-proliferative activity. Highest activity was displayed when the 3^rd position of quinoline was substituted with fluorine containing phenyl sulfonyl urea along with bromine atom in the 6^th position, while a moderate activity was observed with the presence of methyl and chloride containing phenyl substituted urea at the 3^rd position along with bromide substitution at the 6^th position. Moreover, in-silico docking investigations of these compounds against PI3Kγ revealed that a strong hydrogen bond interaction with quinoline ring nitrogen and the amino acid V882 in the hinge region to have crucial influence on the observed activity [70].

Table 1.

In vitro anti-proliferative activity as evaluated by the IC₅₀ values [70].

		IC50 (µM)
R1	R2	HepG-2	A549	PC-3	MCF-7
Pivalonitrile	−F	33.28 ± 1.52	38.52 ± 1.04	66.62 ± 1.28	3.88 ± 0.58
Pivalonitrile	-CH3	31.25 ± 1.50	28.96 ± 1.31	40.59 ± 1.11	3.96 ± 0.59
-OCH3	−Cl	22.56 ± 1.12	30.22 ± 1.23	>100	10.65 ± 0.97
−Br	−Cl	11.13 ± 1.04	71.11 ± 1.85	>100	13.78 ± 1.14
−3-Cl-4-F	−Cl	6.443 ± 0.89	>100	17.72 ± 0.95	6.84 ± 0.83

3.5. NF-κB pathway

Imidazoquinolines were recognized as potent immune response modifiers with proven anticancer activities. These imidazoquinolines were shown to induce apoptosis via various mechanisms that include the activation of the kinase1/c-Jun-N-terminal kinase/p38 pathways and the stimulation of endoplasmic reticulum stress. Their association with the activation of numerous protein kinase signaling pathways, an increase in intracellular calcium release and the cleavage of caspase-4 leads to the degradation of calpain [71]. Furthermore, they stimulate the expression of apoptosis protein inhibitors, along with the stimulation of NF-κB and the accumulation of reactive oxygen species. One such immune response modifier is Imiquimod. Imiquimod is known to influence the TLR-7 and/or TLR-8 by inducing the expression of proinflammatory cytokines and subsequently in the activation of NF-κB signaling [72].

Imiquimod triggers mitochondrial dysfunction, that further leads to the loss of mitochondrial membrane potential, enhance the release of cytochrome c and the activation of caspase-9, caspase-3 and poly(ADP-ribose) polymerase (PARP), etc.

Recent investigations on a series of potent imidazoquinoline derived compounds have revealed their potential to simultaneously contend the pro-inflammatory signaling pathways JAK/STAT and NF-κB [73–76]. Valuable insights into the molecular mechanisms by which imidazoquinolines modulate these critical signaling pathways were reported. These imidazoquinolines have down-regulated the expression of various pro-inflammatory factors that include IL-6, IL-8, IL-1ß, TNF-α, IL-12 and IFN-γ. They have also demonstrated strong anti-inflammatory potential by simultaneously suppressing both JAK/STAT and NF-κB signaling pathways.

3.6. Immunotherapeutics and anti-cancer strategies

Immunotherapeutics in cancer therapies induce the immune system further to raise an immune response against specific tumor cells. Recent approaches to improve the efficacies, to curb the progression and enhance the survival rates, diverse strategies were adopted that include the modulation of immune system, adoptive transfer of immune cells, development of tumor-associated antigen vaccines, etc. With the reported directed enzyme prodrug therapy, it was possible to overcome the severe inflammatory toxicity caused by the systemic routes of administration. In this strategy, an enzyme-directed imidazoquinoline pro-immunostimulant was designed and demonstrated, by utilizing an enzyme – substrate pair to systemically convert the administered prodrug into a cytotoxic payload specifically within the enzyme-rich tumor microenvironment, thereby to trigger the tumoricidal effects. In this approach, a pro-immunostimulant was generated by covalently linking an enzyme substrate to an imidazoquinoline immune-stimulant, to produce a pro-immunostimulant that remains inactive until the immune-stimulant was released via enzyme treatment, to restore its activity.

As shown in Figure 2(c,d) an imidazoquinoline derivative was covalently linked to galactopyranoside that served as a pro-immuno-stimulant and gets converted to active immune-stimulant upon exposure to ß-galactosidase.

However, this strategy was limited by the bystander effect, where the cytotoxic payload cross-reacts with adjacent cells, leading to off-target cytotoxicity (Figure 2(e)). To address this limitation, it was proposed to use enzyme-directed pro-immunostimulants, which would activate bystander immune cells through diffusion of the active immune-stimulants from enzyme-rich cancer cells within the tumor microenvironment (Figure 2(f)).

3.7. Anti-cancer vaccines

Enhancing the immunogenicity of vaccines was considered as a key focus in the development of anti-cancer vaccines. Modulating the immune response was often desired to generate antigen-specific cytotoxicity as well as antibody-dependent cytotoxicity via the activation of natural killer cells and macrophages. Additionally, activating TLR-7/8 activity is another promising approach, as these receptors were found on a wide range of antigen-presenting cells. Numerous small molecule agonists for TLR-7/8 were reported which further project them as an important therapeutic target. However with imidazoquinolines, unfavorable pharmacokinetic profiles along with dose-dependent toxicities were observed that further limits their application for the purpose. It was also found that local administration of imidazoquinolines can result in rapid systemic distribution, thereby triggering systemic inflammatory responses. It was made possible with the covalent linking of imidazoquinolines to large carrier molecules that can prevent uncontrolled systemic dissemination. Recently, the effectiveness of nano-particulate imidazoquinolines in promoting type 1 immune responses was demonstrated through their use as vaccine adjuvants. Though this strategy was proven for the development of human respiratory syncytial virus, it was also extended toward anti-tumor vaccines.

3.8. Antibody-drug conjugates

On the other hand, the technology developed with drug conjugated (ADC) antibodies wherein the tumor-targeting antibodies were used to transport cytotoxins selectively to tumor cells was recently recognized as another effective strategy to treat cancers [77–79]. Though this method was relatively efficient, it lacks meek efficacy and substantial off-target toxicity. To overcome this, the new generation immune-activating therapies have emerged and were successfully demonstrated for a variety of cancers [80,81]. Subsequently, various immuno-stimulatory strategies, including the use of toll-like receptor (TLR) activators, were also developed. Recently, Fang et al. reported another optimization method utilizing ADC technology to deliver a potent series of TLR-7 agonists with high selectivity [82]. To achieve this, a series of imidazo[4,5-c]quinoline agonists (Figure 1(b)) were demonstrated to selectively target the TLR-7 receptors. The design option for 1 and 2 was specifically to improve the permeability while for 3 and 4, it was based on formation of additional hydrogen bonds with the receptor, as reported for Gardiquimod and Resiquimod. This further triggers the human peripheral blood mononuclear cells to release IFNα and influence the antigen-presenting cells toward enhanced production of T-cell activating molecule.

The ADCs developed with a series of imidazoquinoline compounds were obtained by attaching them to an antibody that specifically targets HER2 through a cleavable linker – valine-citruline p-aminobenzyl carbamate. (Figure 1(f)) This led to the development of candidates with high potential and selectivity to effectively activate the TLR-7 pathway in tumor-linked macrophages through “bystander” mechanism. Upon incubation of this ADC with HER2+ cells, it rapidly releases the TLR-7 agonists into the media. Further, the NF-κB pathway was found to be activated when the HER2+ cells were co-cultured with human breast cancer cell lines, to induce the release of IFNα (Figure 2(c,d)). Unlike other reported ADCs wherein Fc-γ-mediated uptake of TLR-7 agonist was observed, in this a passive diffusion into tumor-associated macrophages was observed. Thus this ADC technology was proven to possess significant potentials specifically for oncology applications.

3.9. Structure–activity relationships (SAR) for TLR-7/8 recognition

Preliminary insights into the structural components (Figure 1(e)) involved in TLR-7/8 recognition with precise features responsible for the observed activity and the detailed SAR of various 1 h-imidazo[4,5-c]quinoline were discussed recently. A comprehensive investigation on the chemical space around each reported agonistic scaffolds of TLR-7 and TLR-8 toward the design and development of new candidates was investigated and the EC₅₀ values for the respective derivatives were reported [83]. The significance of NH₂ group at R3 position was realized by its replacement with – NHOH and -NHNH₂ that drastically reduced the TLR-7 activity.

1 h-Imidazo[4,5-c]quinoline derivatives were the first recognized immune stimulators capable of inducing type-I interferons in human cells. Moreover these derivatives were the first to be reported as TLR-7/8 agonists. Using the imidazoquinoline-derived Gardiquimod as the lead structure, a library of 1 h-imidazo[4,5-c]quinoline derivatives with functionalization at the N1, C2 and C4 positions of the imidazoquinoline ring was synthesized. Furthermore, a detailed SAR investigation along with the results of human TLR-7 reporter gene assay revealed their agonistic potentials (Figure 3). Upon careful evaluation, it was observed that the substitution at C2 position was considered as inappropriate site for modification, while the length of C2 substituent was known to significantly influence the TLR-7 activity. Highest activity was observed for C2-n-butyl derivative (14d). While for the C4 substituent, the presence of NH₂ functionality revealed maximum TLR-7 activity. A detailed investigation on the SAR revealed that N1 benzyl-C2-n-butyl analogue 14d to be substantially active and chemically distinct. Moreover, the derivatives 17 and 18 displayed highest activity with EC₅₀ of 8.6 nM and 0.358 μM respectively.

Figure 3.

Library of substituted 1 h-imidazo[4,5-c]quinoline with different functionalization at N1, C2 and C4 position employed for human TLR-7 reporter gene assay.

Table 2 shows the variously functionalized imidazoquinolines at N1, C2 and C4 positions that were tested for TLR-7 activity. This resulted in a focused SAR study around Gardiquimod that led to the identification of a highly active and chemically distinct N1-benzyl-C2-n-butyl derivative. Highly potent and active compounds were produced when the substituent groups at N1 and C2 positions were swapped on the lead molecule to yield 17 and 18. The significant role of imidazole ring system toward recognizing ligands by TLR was revealed when completely inactive triazole and cyclic urea compounds were obtained by replacing the imidazole ring with the respective moieties. The summary of various substituents on the imidazoquinoline ring and the exerted activities is shown in Table 2.

Table 2.

Activity based on human TLR-7 reporter gene assay upon different functionalization at the N1, C2 and C4 positions.

Active	Partially active	Inactive
C-2 substituent
Based on the nature of amines
	N-ethyl (20a) N-ethylenenitrile (20b)	N-propylene amino (20c) N-triethyleneglycol (20d) Guanidine (20e) Acyl (20f, 20 g) Thiourea adduct (20i) n-undecyl group (20 h)
Based on the length of alkyl chain
n-butyl derivative (21d) C2-n-pentyl derivative (21e)		methyl (21a) ethyl (21b) n-propyl (21c) n-hexyl (21f) n-heptyl (21 g) n-nonyl (21 h)
Functionalization with an unsaturated hydrocarbon chain
	n-butyl-3-ene (21 l) n-butyl-3-yne (21k)
Based on lipophilicity
		n-undecane (21i), n-pentadecane (21j) n-undecylamino)methyl (20 h)
C-4 substituent
4-NH2 (21d)	4-NHOH (23e) 4-NHNH2 (23f)	4-Cl (23a), 4-OH (23b) 4-Ph (23c) des-amino analogue (23d)
N-1 substituent
Amino-methyl Substituted benzyl analogues	Naphthalene Biphenyl

Similarly, C7 derivatization of imidazoquinoline was investigated by Hunt et al. (Figure 4(a)) [84]. A variety of electron-withdrawing and electron-donating groups were incorporated at the C7 position of the imidazoquinoline scaffold. Having 2-hydroxy-2-methylpropyl substituent at N1 position and ethoxymethyl moiety at C2 position, various analogs including C7-methoxy, hydroxyl, nitrile, chloro, etc was synthesized. Moreover the presence of electron-donating groups has enhanced the activity for TLR-7/8 compared to electron-withdrawing groups. The presence of C7-hydroxy substituted compound was the most potent TLR-7 agonist while no significant TLR-8 activity was observed under similar conditions. Moreover, efforts were taken to investigate the SAR for the derived structures of imidazoquinolines functionalized at N1 position on the C7-methoxycarbonyl derivatized imidazoquinolines (Figure 4(b)).

Figure 4.

(a) Derived structures of imidazoquinolines functionalized at C7 position. (b) Derived structures of imidazoquinolines functionalized at N-1 position on the C-7-methoxycarbonyl imidazoquinolines that were tested for h-TLR-7/8 activity.

Upon investigating the SAR of C7-methoxycarbonyl imidazoquinolines for TLR-7 and TLR-8 activities, enhanced substituent tolerance was noted for TLR-7 as compared to TLR-8. It was also observed that substitution with C2-n-butyl to possess maximum TLR-7 than other shorter chain length analogs while TLR-8 activity was observed only for C2-n-butyl (19d) and C2-n-pentyl (19e) analogues. As in the case of 26e, the loss of the hydroxyl group at the N1 substituent significantly influences the TLR-8 activity.

In summary, the imidazoquinoline scaffold was systematically investigated by examining the impact of substitutions at four critical positions: C2, C4, N1 and C7. The presence of substituents like a free amine group at the C4 position and n-butyl group at the C2 position was found to be most favorable for the observed TLR-7/8 agonistic activity. Moreover, enhanced TLR-7/8 activity was observed when substituents with basic functional moieties were introduced at N1 and electron-donating groups at C7 positions.

3.10. Modulation of imidazoquinoline trafficking for rational designing of immune-therapeutics

In most solid tumors the acquired multidrug resistance (MDR) is associated with the overexpression of P-glycoprotein (P−gp). Cancer cells with MDR are associated with the identification of over-expressed P-gp in the plasma membrane that subsequently recognizes other transporters related with drug efflux, like multidrug resistance-associated protein 1 (MRP1) and multixenobiotic resistance (MXR). The major issue observed with multidrug-resistant cancers is their attenuated chemotherapeutic efficacy induced via drug efflux mechanism wherein the drugs are transported from the cells to the extracellular space with the help of ABC (ATP-Binding Cassette) transporters like P-gp. The antitumor drug resistance was mainly driven by increased activity of drug efflux pump like ABC superfamily, reduced influx of drugs, activated DNA repair machineries, detoxification and influenced expression of apoptosis-associated protein Bcl-2 and tumor suppressor protein p53.Therefore, the intracellular concentration of cytotoxins can be reduced in the cells by the expressed P−gp under physiological conditions [85]. The mechanism of MDR associated with influenced drug efflux in tumor cells is summarized in Figure 5(c). On the other hand, immunotherapies with toll-like receptor agonists are known to affect the immune cells’ tumor-infiltrating activity and augment the drug efflux mechanism in MDR cancers. Until recently, the precise mechanism underlying the developed drug resistance related to TLR agonist efflux remained unidentified. Further to investigate this, the P-gp-mediated efflux activity of TLR-7/8 agonists (22) was examined (Figure 5(a)). The findings indicate that the observed increase in efflux susceptibility may be a critical factor toward the strategic design of next-generation immunotherapies that aim toward controlling the activity of tumor-infiltrating immune cells. A comparison of the P-gp substrates Imiquimod, Resiquimod and Gardiquimod in relation to P-gp-mediated efflux across multidrug-resistant cancer cell lines was investigated.

Figure 5.

(a) Enhanced P-gp mediated efflux observed in the case of MDR cancer cells. (b) Schematic representing the anti-MMR Nb-IMDQ conjugates (IMDQ: imidazoquinoline-based TLR-7/8 agonist 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine). (c) Structures of modified imidazoquinolines at the N-1- and C-2-positions with favorable structure-activity relationship. (d) Structures of various imidazoquinolines with proven interferon inducing activity that preceded the investigations on TLR.

3.11. Imidazoquinoline-based nanotechnology - imidazoquinoline coupled nano-bodies

On the other hand, nano-bodies (single chain antibody fragments) coupled with an imidazoquinoline (Figure 5(b)) revealed to be a promising TLR-7/8 agonist [86].

This imidazoquinoline possessed the ability to convert pro-tumoral macrophages to their pro-inflammatory state with efficient drug delivery to the target site (macrophage mannose receptor), inducing rigorous suppression of tumor growth and also to promote the anti-tumor T-cell responses. Unlike the previously reported nano-bodies, its pharmacokinetic profiles did not reveal any systemic inflammation due to its site specific and quantitative coupling.

The abundant expression of TLR-7 and TLR-8 proteins in antigen-presenting cells projects them as promising adjuvant candidates for developing novel and effective anti-tumor vaccines. In line with this significance, a new series of imidazoquinoline derivatives was developed, featuring N-isobutyl functional group in the imidazole moiety, like in the case of imiquimod, along with a 1,2,3-triazolyl group. The target specificity of all the novel analogues was assessed with TLR-7/8 transfected cell lines, which revealed that the observed activity of TLR-7/8 was primarily influenced by the nature and position of the substituent groups, as well as the chain length of the alkyl substituents [87].

Recent studies have demonstrated that combining an imidazoquinoline-based TLR-7/8 agonist with a monoclonal antibody enhances the antibody-dependent cellular cytotoxicity (ADCC) [88]. Additionally, several small-molecule TLR-7/8 agonists were found to elicit higher cytokine levels compared to the standard imiquimod. In combination with monoclonal antibody therapy, these agonists were further assessed for their ability to enhance ADCC, leading to the secretion of pro-inflammatory cytokines and the activation of natural killer cells. Further, these monoclonal antibodies also displayed enhanced anti-cancer activity. Furthermore, the creation of adaptive immunity at an early stage was also observed through the stimulation of CD-8 T cells. The potential to design TLR-7/8 agonists that can modulate cytokine profiles, enhance the activation of natural killer cells and ADCC upon combining the above with anti-EGFR monoclonal antibody was demonstrated.

Recently, antibody drug conjugates (ADCs) with tumor-targeting antibodies were reported to selectively deliver potent cytotoxins to tumor tissues [78]. However, this technology is constrained by its relatively low efficacy and substantial off-target toxicity. Further to overcome this limitation, new therapies that involve immune-activation were reported and demonstrated by the PD-1/PD-L1 (programmed cell death protein/programmed cell death ligand) inhibitors for a range of cancers. This prompted the investigation of new immune-stimulant strategies that include toll-like receptor activators. Employing this ADC technology, the targeted delivery of a potent series of TLR-7 agonists was demonstrated wherein a series of imidazo[4,5-c]quinoline derivatives as TLR-7 agonists was reported to release IFNα and up-regulate the CD-86 on antigen-presenting cells.

Efforts to modify imidazoquinolines aimed toward understanding the structure-activity relationship (SAR) at the N-1 and C-2 positions that further led to the development of several drugs, including imiquimod, resiquimod and many other potent analogues as shown in Figure 5(c).

Recently it was demonstrated that TLR-7/8 can tolerate a variety of substituents at the C-7-position at the cost of increased potency and modified cytokine profiles. The most notable TLR-7/8 agonists that revealed multiple fold enhanced activity than resiquimod for TLR-8 and/or TLR-7 and imiquimod for TLR-7 were developed. The agonist activities of various analogs are listed in Table 3.

Table 3.

Activities of various N-1, C-2 and C-7 substituted imiquimod analogs.

					EC50 (µM) ± SD*
		R1	R2	R3	TLR7	TLR8
	1 2 3 4 5 6 7 8 9 10 11	OMe Cl Cl Cl Cl CN CN CN CN OH	H OH H H OH OH H H OH OH H	n-Bu n-Bu n-Bu CH2OC2H6 n-Bu CH2OC2H6 n-Bu CH2OC2H6 n-Bu CH2OC2H6 n-Bu	0.11 ± 0.01 0.081 ± 0.009 0.3 ± 0.02 0.91 ± 0.09 0.084 ± 0.007 0.63 ± 0.03 0.99 ± 0.40 6.8 ± 0.7 0.79 ± 0.23 3.5 ± 0.7 0.073 ± 0.005	>100 1.7 ± 0.3 >100 >100 1.5 ± 0.2 4.9 ± 0.6 >100 >100 3.9 ± 0.2 27 ± 6 >100

* Average EC₅₀ values were determined using either hTLR-7/-8 transfected HEK-Blue cells along with corresponding Null controls.

Investigations on the demonstrated TLR-7/8 agonist active small heterocyclic molecules like imiquimod, resiquimod, gardiquimod, CL097, CL075, 3 M–003, etc., have revealed that modest structural changes resulted in major variations in their activities (Figure 5(d)) [89]. The electronic configuration on the heterocyclic systems was known to significantly influence the observed agonistic activity [38]. An elaborate SAR for a variety of chemotypes derived from the imidazoquinoline-based scaffold was recently investigated [83]. Moreover, these TLR-7/8 agonists serve as promising candidates for vaccine adjuvants and further research is focused toward identifying more potent and suitable candidates for drug discovery.

The synthetic imidazoquinoline derivative CL091 displayed the ability to induce the production of interferon via the activation of TLR-7 and TLR-8 that further activates the downstream signaling pathways to produce type I interferons (IFN-α and IFN-β) and pro-inflammatory cytokines like., TNF-α, IL-6, etc. The strong interferon inducing characteristic of CL091 was utilized in the investigation of cancer immunotherapy. CL075 was known to primarily act as TLR-8 agonist with moderate activity on TLR-7 as this drug stimulates IFN-β while displaying weaker effect on IFN-α. It was also known to induce less production of IFN-α as compared to other imidazoquinolines like imiquimod and resiquimod. Its ability to stimulate innate immunity was investigated for its role in cancer immunotherapy. On the other hand, 3 M–003 was known to activate both TLR-7 and TLR-8 further to trigger immune response. It was investigated by several researchers for its ability to boost the immune system’s ability to recognize tumor cells.

The immune-stimulatory activity observed in these analogues was attributed to the stimulation of type 1 interferons (IFN-α and β), even though the precise mechanism of action remained unclear until the TLRs were identified and reported. Research has identified molecules that can induce interferons by activating the TLRs [90].

3.12. Synthesis of important imidazoquinoline-derived anti-cancer agents

Till date there exist several challenges toward the development of effective methodologies for synthesizing combinatorial libraries of molecules in drug discovery research. Numerous strategies including the solution- and solid-phase synthetic approaches, deconvolution methods, etc., were developed. Recently, microwave assisted combinatorial synthesis has attracted the attention of many researchers, as they are efficient, quick and yield cleaner products as compared to other conventional methods. This technology was recently extended for drug-discovery applications as an effective tool. Some of the synthetic approaches to obtain potential pharmacological molecules are discussed.

Synthesis of variously derived 3-chloro-4-arylsubstituted-1-[(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)amino]azetidin-2-ones at the 4-position of imidazoquinoline as potent anticancer agents via β-tubulin inhibitory activity was reported recently [91].

Scheme S1 (supplementary information) depicts the synthetic scheme for target compounds. Previously reported procedures were employed to achieve (4-chloro-1-isobutyl-1 h-imidazo[4,5-c]quinoline from 2,4 dihydroxyquinoline. The product mentioned above was then treated with hydrazine hydrate to produce 4-hydrazino-1-isobutyl-1 h-imidazo[4,5-c]quinoline. This compound was subsequently reacted with various substituted aromatic aldehydes in ethanol to yield a series of hydrazones, specifically 4-(substituted arylidenyliminoamino)-1-isobutyl-1H-imidazo[4,5-c]quinolines. The imidazo[4,5-c]quinoline derivatives containing triethylamine were then treated with chloroacetyl chloride to obtain the corresponding 3-chloro-4-arylsubstituted-1-[(1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)amino]azetidin-2-ones. (30).

Most of the derivatives exhibited very good anticancer activity when evaluated in HeLa cells. An enhanced anti-tumor activity was observed when the imidazoquinoline moiety was linked with biphenyl group via azomethine linker. Also it was observed that the activity was enhanced for the cyclized analogues as compared to uncyclized derivatives.

Recently imidazoquinoline based TLR-7/8 adjuvants were synthesized and revealed to have enhanced efficacy [87]. This novel imidazoquinoline derivative, featuring an N-isobutyl substitution on the imidazole ring and a 1,2,3-triazolyl moiety attached to the alkyl group at the imidazolemethyne carbon, was synthesized via triazolyl click chemistry. (see Scheme S2, supplementary information) The evaluation of immunomodulatory properties of these analogues revealed their ability to target the TLR-7 receptors along with pro-inflammatory immune response. It was believed that the bioavailability and interaction potential of these 1,2,3-triazoles with biomolecular targets to be enhanced through hydrogen bonds that further increase the solubility of imdiazoquinoline moiety. These derivatives have exhibited potent adjuvant activity and target specificity when assessed by their TLR-7/8 agonist activity. Moreover, these novel imidazoquinoline derivatives exhibited potent immune-modulating properties via TLR-7/8 binding interactions.

Using a single-pot microwave-assisted method, our group had reported the synthesis of novel biologically significant pyrido-fused imidazo[4,5-c]quinoline derivatives [49,92]. These were reported as privileged scaffolds with broad scope pharmacological properties including their inhibition potential against PI3K/PKB pathways, TNFα inhibitor, etc. The ability of these derivatives to bind to the active site of PI3K was assessed, along with their corresponding anti-cancer activity. Similarly several drugs in clinical trials incorporate the privileged scaffolds that have revealed high affinity toward binding to multiple biological targets. Privileged scaffolds serve as core structures for developing bioactive molecules and hence were being widely used in drug design. A recent review has revealed that among various privileged natural product scaffolds, the substantial presence of benzopyrone class of compounds were reported among numerous biologically active compounds. Numerous benzopyrone based small molecules are under clinical research.

Similarly, pyrido-fused imidazo[4,5-c]quinolines were synthesized following a rapid one-pot microwave-assisted sequential method via the Pictet – Spengler cyclization approach (see Scheme S3 Supporting information). Computational investigations have revealed the interaction pattern (Figure S1, supplementary information) of these compounds with PI3K, an important enzyme that has attracted therapeutic investigations toward the treatment of cancer [49]. In particular inhibition of PI3K signaling pathway was reported as one of the effective strategies in the development of cancer therapeutics [93]. Investigations have revealed that the stabilization was majorly contributed from van der Waals interaction energy. The occurrence of stabilizing intermolecular hydrogen bonding interaction within the binding pocket of PI3K is also shown in Figure S1, supplementary information.

It was well established that hydrogen bonds not only stabilize the interaction of ligands with bio-molecules but also assist in maintaining their structural stability. Since the proteins undergo continuous change in their 3-dimensional structure at different stages of their cellular functions, it was believed that even minor structural alterations may greatly influence their functionality. These studies have provided evidence supporting the consideration of suitable pyrido-fused imidazo[4,5-c]quinoline compounds as potential anti-tumor agents.

4. Conclusions

Imidazoquinolines represent a significant class of heterocyclic compounds with established potential in pharmaceuticals and therapeutics. This review explores the role of imidazoquinoline derivatives as anticancer agents. They play a crucial role in regulatory and immune signaling pathways, including the modulation of immune-related cytokines. Additionally, they are known to target various overexpressed biomarkers in cancer cells. HER2 is one such transmembrane tyrosine kinase belonging to EGFR family of proteins that was utilized in the development of nucleic acid based vaccines in association with the well-known immune response modulators including imidazoquinoline derivatives. These compounds further enhanced the production of endogenous cytokines, leading to the activation of TLR-7/8. Their role as adjuvants in various vaccines is well established. Given the challenges of poor efficacy and off-target cytotoxicity in antibody-drug conjugates (ADCs), a new generation of immune-activating therapies utilizing imidazoquinoline-derived immunostimulants were successfully developed. Recently, ADCs containing HER2-specific antibodies with a cleavable linker were reported to follow a bystander mechanism, activating the NF-κB pathway and promoting IFNα release. Comprehensive studies on the structure-activity relationships of imidazoquinolines as TLR agonists have demonstrated that various substituents at the N-1, C-2, and C-7 positions were well tolerated, leading to enhanced potency and modified cytokine profiles. Imidazoquinolines were also known to influence the activity of tumor-infiltrating immune cells in their vicinity and enhance drug efflux in multidrug-resistant (MDR) tumors. The ability to modulate payload delivery within tumor cells has been successfully demonstrated in enzyme prodrug therapy, where an enzyme-activated imidazoquinoline pro-immunostimulant was employed.

With all these developments, there still exists the gap for the efficiency of these drugs to be enhanced with possibilities to overcome the existing limitations with the marketed anticancer agents. Though the imidazoquinoline-based anticancer drugs have demonstrated their ability to activate toll-like receptors that can lead to antitumor immune responses, their clinical application faces several limitations. The poor solubility and stability under biological microenvironment result in suboptimal pharmacokinetic properties with limited bioavailability. Also the activation of TLRs is known to produce unintended immune responses that can trigger off-target drug effects. The poor understanding on the resistance mechanisms developed through various pathways limits the efficacy of imidazoquinoline-based therapies. All these concerns need to be addressed and new leads for synthesis, structure-activity relationship investigations, biological activities of new preclinical imidazoquinolines need to be researched. By overcoming these limitations it is possible successful integration of imidazoquinoline-based therapies into clinical oncology.

5. Future perspectives

Despite recent advancements, imidazoquinoline-based anticancer drugs face challenges in clinical application, including poor solubility, stability and bioavailability, leading to suboptimal pharmacokinetics. While these drugs activate toll-like receptors (TLRs) to trigger antitumor immune responses, unintended immune effects and off-target activity remain as major concerns. Additionally, resistance mechanisms through various pathways limit their efficacy. Addressing these issues requires further research into synthesis, structure-activity relationships and biological activities of new preclinical imidazoquinolines. Overcoming these limitations could be the focus of research on tumor drug discovery.

Funding Statement

This work was not funded.

Article highlights

HER2 as target

• Nucleic acid vaccines targeting HER2 are more effective toward inducing immune responses. Further tuning of activity enhancement is possible through induced immune-modulatory cytokines.

• Advanced combination therapies include sonodynamic and photothermal methods along with TLR agonists. These molecules also serve as vaccine adjuvants.

Antibody-drug conjugates

• In the development of antibody-drug conjugates, most of the reported imidazoquinoline-based drugs were found incompatible with generic linkers.

Opioid Growth Factor Receptor as target

• Understanding the mechanism of action of reported imidazoquinoline-based drugs are known to enhance the growth inhibitory effect OGF-ORFr complexes without producing proinflammatory effects.

Targetting NF-κB Pathway

• Imiquimod, influences the TLR-7 and TLR-8 by enhancing the production of proinflammatory cytokines and further toward activating the NF-κB signaling.

Immunotherapeutics and Anti-Cancer Strategies

• The conversion of prodrug into cytotoxic payload to trigger tumoricidal effects through enzyme linked immunostimulant assay was limited by the bystander effect.

Anti-Cancer Vaccines

• Most of the reported imidazoquinoline drugs display unfavorable pharmacokinetic profiles with dose dependent toxicities.

• Several synthetic strategies were adopted following the structure-activity relationship of the reported drugs. In many cases, the observed activity was significantly influenced by the nature and position of the substituents in the imidazoquinoline scaffolds.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17568919.2025.2491303