Quinones of various chemical families have biological activities [1,2,3], and both natural and synthetic quinones are widely used as drugs [4,5]. Anthracyclines, a large group of quinonoid compounds produced by different strains of Streptomyces, exert antibiotic and antineoplastic effects, and are used to treat some forms of cancer [5]. The best-known members of this family are daunorubicin and doxorubicin, the first identified anthracyclines [6]. Mitomycin C and streptonigrin produced by Streptomyces and the synthetic epirubicin and mitoxantrone are used as anticancer drugs [7]. Although these and other quinonoid compounds are effective in the treatment of many different forms of cancer, their side effects—the most severe being cumulative heart toxicity—limit their use [8,9,10]. The development of quinonoid compounds that display antineoplastic activity, but are less toxic, is a major therapeutic goal.

A large number of cannabinoids have been synthesized and tested in various in vitro and in vivo assays, including models of several diseases [11,12,13,14]. A cannabinoid anticancer p-quinone, HU-331 (HU standing for Hebrew University), was synthesized by our group back in 1968, to address a chemical question of which cannabinoids give a purple color when hashish extract is treated by 5% aqueous KOH in methanol (the Beam Test) [15]. Much later, in the 1990s, this compound was suggested to be a hepatic metabolite of CBD [16,17]. It was also found to decrease the microsomal content of a monooxygenase CYP450 enzyme, an effect prevented by co-incubation with glutathione (GSH) or cysteine, presumably by the radical-scavenging action of these antioxidants [16]. In 2001, while searching for possible anticancer cannabinoids, we synthesized HU-331 again, this time as an attempt to combine the quinonoid anticancer moiety with cannabinoid molecules, which are mostly non-toxic. Cannabinoids are known to possess distinct pharmacokinetic and distribution patterns than the known quinonoid anticancer drugs. HU-331 was shown to have very high efficacy against human cancer cell lines in-vitro and against in-vivo grafts of human tumors in nude mice, especially in colon carcinoma HT-29 and in Raji lymphoma [18]. The mechanism of action of HU-331 in cancer cells was found to be the inhibition of topoisomerase IIα, without topoisomerase poisoning [19]. It caused neither cancer cells apoptosis nor cell cycle arrest. It did not affect the intracellular pathways of p38MAPK, pERK, c-jun or p53.

The effect of HU-331 as inhibitor of topoisomerase IIα seemed very selective [19]. This finding was later also supported by Dewesee’s group [20] and Marson’s group [21]. In addition, we found it to possess anti-angiogenic effects [22], and unlike most other quinones, HU-331 was not cardiotoxic [23,24]. However, it seems that in different cells the mechanism of action of HU-331 may vary from the one found by us. The first mention of a different mechanism was by Usami et al. in 2008, who suggested that HU-331 generated reactive oxygen species (ROS) during mouse hepatic microsomal metabolism in the presence of NADPH [25]. Another mechanism was suggested by Wu and Jan (2010), who found that HU-331 treatment significantly enhanced splenocyte apoptosis in a time- and concentration-dependent manner. A gradual decrease in the levels of cellular thiols and glutathione was detected in HU-331-treated splenocytes. The apoptosis and thiol diminishment induced by HU-331 were abrogated in the presence of thiol antioxidants, including N-acetyl-L-cysteine and N-(2-mercaptopropionyl) glycine, while the non-thiol antioxidants catalase and pyruvate were ineffective [26]. The search for possible additional mechanisms of HU-331 still continues, with a recent publication showing that HU-331 modulates a peroxisome proliferator-activated receptor gamma (PPARγ)-based activity [27].

After our reports on HU-331, several groups reported syntheses of novel cannabinoid-like quinones. In 2013, Petronzi et al. synthesized some novel quinones, related to HU-331. In the most potent molecule of this series (Compound 1, Figure 1), the side-chain is elongated to six carbon atoms (instead of five carbon atoms as in HU-331), and is shifted to position 2 of the quinone ring, leaving position 3 open. The terpene ring that is present in position 6 of HU-331 is deleted. This molecule was found to be active against some cancer cell lines, especially M14 melanoma. The mechanism of action of this new molecule differs from that of HU-331: a time-dependent pro-apoptotic activity on human melanoma M14 cell line was seen, mediated by ROS, caspases activation and poly-(ADP-ribose)-polymerase (PARP) protein cleavage. Neither topoisomerase I nor II were involved [28].

That same year, Jagerovic’s group synthesized p-quinones from their previously-discovered cannabinoid-like chromenopyrazoles [29]. Some of them showed significant affinity in the sub-micromolar range to both CB1 and CB2 receptors. The most active compound was named PM49 (Compound 2). These compounds were more effective in prostate carcinoma than in hepatocellular carcinoma cells. The mechanism of action of PM49, however, differed from that of HU-331. The CB1 antagonist rimonabant, as well as the PPARγ antagonist GW 9662 and the antioxidant N-acetyl cysteine (NAC), were able to significantly reduce the inhibition of cell viability induced by PM49. It was found to cause cancer cells apoptosis, also unlike HU-331 [30]. Later (in 2015), the same group continued their research on compounds related to the chromenopyrazoles quinones, but in the ortho configuration. All the novel cannabinoid quinone derivatives displayed potent growth inhibitory effects on triple-negative MDA-MB-231 cells with low micromolar IC50 values. Compound 3, selected for further development, was also able to effectively reduce the growth of triple-negative breast cancer xenografts in vivo [30].

In 2018 ElSohly’s group reported research with cannabinoid anticancer quinones based on tricyclic THC-like cannabinoids. The introduction of the p-quinone moiety by photooxygenation of THC and its derivatives led to a loss of affinity to the cannabinoid receptors CB1 and CB2. The quinone derivatives exhibited anticancer and marked antimicrobial activity. One of these (Compound 4) was a potent anti-cryptococcal agent. The best agent against S. aureus was Compound 5; both compounds were antiproliferative in different cancer cell lines, while others showed anti-candidal activity [31].

Recently, Marson’s group added an additional piece to the puzzle of cannabinoid quinones and their action on topoisomerase II, by synthesizing about 15 derivatives related to HU-331, and assaying them for topoisomerase inhibition and cancer cell growth inhibition (mostly DU-145 prostate carcinoma) [21]. The most active derivatives were those with cyclohexyl and cyclopentyl substitution at the place of limonene in HU-331, with no substituent in position 2 of the quinone ring, and with pentyl or methyl groups in position 3 (Compounds 6 and 7).

Casares et al. recently published that the methoxylated HU-331 derivative has dual activity as NRF2 activator and BACH1 inhibitor [32]. The axis NRF2-BACH1 has anti-inflammatory and antioxidant properties that could be exploited pharmacologically to obtain neuroprotective effects. Indeed, this derivative provides neuroprotection in cell models of relevance to Huntington’s disease [32].

Recently, a Polish group investigated the anti-cancer activity of several topoisomerase inhibitors, amongst them HU-331, combined with cisplatin. All the investigated topoisomerase II inhibitors in combination with cisplatin efficiently killed U-87 cells. The most potent cytotoxic effect was observed for cisplatin with HU-331 [33].

In a different line of cannabinoid quinone research, back in 2012, researchers in the VivaCell company, in collaboration with colleagues in two Spanish universities, searched for additional biological activities, different from cancer cell growth inhibition. The quinone of cannabigerol, named VCE-003 (Compound 8), showed prominent anti-inflammatory activity, protected neuronal cells from excitotoxicity, activated PPARγ transcriptional activity and inhibited the release of pro-inflammatory mediators in LPS-stimulated microglial cells [34]. It also ameliorated the symptoms associated with TMEV (Theiler’s murine encephalomyelitis virus) infection, a model of multiple sclerosis (MS), leading the authors to assume that cannabinoid quinones may be useful in the treatment of MS and other neuroinflammatory disorders [34]. In a later experiment (in 2014), the same group showed that VCE-003 ameliorated the neurological defects and the severity of MOG-induced EAE (myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis) in mice through CB2 and PPARγ receptor activation, at least partially, as antagonists of both these receptors partially blocked VCE-003 activity [35]. Later, in 2016, this compound was modified to VCE-003.2 (Compound 9), which is a representative of a second generation of cannabigerol quinone non-electrophilic derivatives, possessing the same anti-inflammatory and neuroprotective effect [36]. In line with the authors’ initial observation of the action of cannabinoid quinones in neurodegenerative diseases, they assayed VCE-003.2 in Huntington’s models of striatal neurodegeneration induced by quinolinic acid and 3-nitropropionic acid. VCE-003.2 prevented medium spiny DARPP32+ neuronal loss in these Huntington’s-like disease mice models, improving motor deficits, reactive astrogliosis and microglial activation, inhibiting the upregulation of proinflammatory markers and improving antioxidant defenses in the brain [36]. In further work on this compound, in 2018, it was also shown to protect the mice against Parkinson’s disease development. The administration of VCE-003.2 to lipopolysaccharide (LPS)-lesioned mice (a model for Parkinson’s disease) attenuated the loss of tyrosine hydroxylase (TH)-containing nigrostriatal neurons, and in particular, the intense microgliosis caused by LPS in the substantia nigra [37]. In a very recent study (2021), VCE-003.2, as well as cannabigerolic acid quinone and its sodium salt, were found to be active in 6-OHDA lesioned mice, an additional model of Parkinson’s disease [38].

The analysis by quantitative Polymerase Chain Reaction (qPCR) of proinflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) in the striatum showed that they were markedly elevated by the LPS lesion, and strongly reduced by treatment with VCE-003.2. The effects of VCE-003.2 in LPS-lesioned mice implied the activation of PPARγ receptors, as they were attenuated when VCE-003.2 was co-administered with a PPARγ inhibitor [37]. However, the in-vitro effects were not attenuated by PPARγ inhibitors, suggesting additional possible mechanisms [37]. Oral administration of VCE-003.2 also generally helped in a Parkinson’s model, though in higher doses than the previous intraperitoneal treatment [39]. In an additional neurodegenerative disease model with SOD1G93A mutant mice (a model of Amyotrophic Lateral Sclerosis, ALS), treatment with VCE-003.2 improved most of the neuropathological signs such as weight loss and neurological deterioration, which was associated with a marked loss of spinal cholinergic motor neurons and glial reactivity, thus indicating the effectiveness of this compound also for ALS treatment [40]. This molecule, which is being developed by Emerald Health Pharmaceuticals, is now named EHP-102, and was granted both FDA and EMA orphan designation for the treatment of Huntington’s disease.

In parallel with CBG-quinones derivatives, the same research group worked also on CBD-quinones. VCE-004.8, a benzyl-amino derivative of HU-331 (Compound 10), was found to possess a dual agonist activity on PPARγ and CB2 receptors, and anti-fibrotic activity in scleroderma models [41]. Another derivative, VCE-004.3, a pentyl-aminoquinone derivative of HU-331 (Compound 11) showed the same activity [42]; however, the company apparently preferred to move forward with the VCE-004.8 derivative. It was reported to attenuate adipogenesis, to prevent diet-induced obesity [43], to activate the hypoxia inducible factor (HIF) pathway, to prevent demyelination, axonal damage, immune cell infiltration in EAE and to be effective in TMEV models of MS [44]. Furthermore, VCE-004.8 was formulated for oral administration, the anti-fibrotic activity of which was the same as an intraperitoneal injection [45]. In a recent publication (2020), this oral formulation was also found to be active in the MS models [46]. Now this molecule, which is being developed by Emerald Health Pharmaceuticals, is named EHP-101, and was granted both FDA and EMA orphan designation for the treatment of scleroderma. At present, it is in Phase II clinical trials on Diffuse Cutaneous Systemic Sclerosis, a subtype of systemic scleroderma.

The structure-activity relationship (SAR) of the cannabinoid quinones is still not very clear. Different molecules possess different mechanisms of action, especially those of the original HU-331 line, and those in which position 2 is substituted with amine derivatives.

Now we report the syntheses of additional compounds in the cannabinoid quinone series, which presumably may help to shed more light on the SAR and the mechanism of action of HU-331 and related compounds.