Saturated Cannabinoids: Update on Synthesis Strategies and Biological Studies of These Emerging Cannabinoid Analogs

Natural and non-natural hexahydrocannabinols (HHC) were first described in 1940 by Adam and in late 2021 arose on the drug market in the United States and in some European countries. A background on the discovery, synthesis, and pharmacology studies of hydrogenated and saturated cannabinoids is described. This is harmonized with a summary and comparison of the cannabinoid receptor affinities of various classical, hybrid, and non-classical saturated cannabinoids. A discussion of structure–activity relationships with the four different pharmacophores found in the cannabinoid scaffold is added to this review. According to laboratory studies in vitro, and in several animal species in vivo, HHC is reported to have broadly similar effects to Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive substance in cannabis, as demonstrated both in vitro and in several animal species in vivo. However, the effects of HHC treatment have not been studied in humans, and thus a biological profile has not been established.


Introduction
Cannabis and cannabis substituents have been used in medicine within the United States for centuries and were first described in the United States Pharmacopeia in late 1850 [1]. Due to legal ramifications and political duress, cannabis was dropped from the United States Pharmacopeia in the 1940s and labeled a controlled substance in the 1970s. These bureaucratic changes have limited advancements within the field of cannabinoid chemistry [2]. The first cannabinoid was not elucidated until the 1940s, when cannabidiol (CBD) was identified, followed by cannabinol (CBN) [3]. As cannabinoid research becomes accessible again, novel and rare cannabinoids have been elucidated through modern analytical techniques, garnering attention, and popularity. However, knowledge about these cannabinoids remains limited to non-existent. Cannabinoid research as a whole has primarily focused on the safety and efficacy of CBD and THC (tetrahydrocannabinol) for specific ailments and has largely ignored the hundreds of other currently identified cannabinoids that Cannabis sativa biosynthesizes in various concentrations [1][2][3][4]. The primary focus of cannabinoid chemistry and the multitude of studies that have been performed are mostly on CBD, and THC, evaluating their safety and effects on certain ailments including but not limited to inflammation and anti-proliferative/pro-apoptotic effects within the body [5].
Lee [25] extended the method to synthesize a wide group of hexahydrocannabinol derivatives using several types of resorcinols and naphthols. As seen in Table 1, the cycloaddition reactions were accomplished with resorcinols, including ester groups on the benzene ring and with 1-and 2-naphthol. Lee [25] employed the same hetero Diels-Alder approach, for the synthesis of (R) HHC (1) and (S) HHC (7), but he used ethylenediamine diacetate (EDDA) (20 mol %) as a catalyst in the presence of triethylamine (TEA) instead of Et 2 AlCl. The reaction mixture was refluxed in xylene for 24 h to afford (9R)-HHC (1) and (9S)-HHC (7) with a 72% and 73% yield, respectively.
Lee [25] extended the method to synthesize a wide group of hexahydrocannabinol derivatives using several types of resorcinols and naphthols. As seen in Table 1, the cycloaddition reactions were accomplished with resorcinols, including ester groups on the benzene ring and with 1-and 2-naphthol.   Compounds 21, 22, and 26 were obtained with higher yields than presence of a carbonyl group in the ortho-position related to one of the on the phenyl ring. This fact can be elucidated due to the hydrogen b hydroxyl group and the carbonyl group of the ethyl ester con Compounds 21, 22, and 26 were obtained with higher yields than 20 and 25 for presence of a carbonyl group in the ortho-position related to one of the hydroxyl grou on the phenyl ring. This fact can be elucidated due to the hydrogen bond between hydroxyl group and the carbonyl group of the ethyl ester conferring a hig Compounds 21, 22, and 26 were obtained with higher yields than presence of a carbonyl group in the ortho-position related to one of the on the phenyl ring. This fact can be elucidated due to the hydrogen b hydroxyl group and the carbonyl group of the ethyl ester con Compounds 21, 22, and 26 were obtained with higher yields than 20 and 25 for presence of a carbonyl group in the ortho-position related to one of the hydroxyl grou on the phenyl ring. This fact can be elucidated due to the hydrogen bond between hydroxyl group and the carbonyl group of the ethyl ester conferring a hig 75 a Reaction conditions: starting material (1.0 mmol), citronellal (1.5 mmol), EDDA (20% mol), TEA (0.2% mol) in xylene [21]. Compounds 21, 22, and 26 were obtained with higher yields than 20 and 25 for the presence of a carbonyl group in the ortho-position related to one of the hydroxyl groups on the phenyl ring. This fact can be elucidated due to the hydrogen bond between the hydroxyl group and the carbonyl group of the ethyl ester conferring a higher regiospecificity to the cyclization reaction, which is likely to occur at the position without hydrogen bonding. On the other hand, the stereospecificity during the intramolecular Diels-Alder reaction could be explained considering that in the transition state (21a), the methyl group adopted a coplanar structure in the chair configuration, so the exo-transition state is energetically more favorable than the endo-transition state, as shown in Scheme 3.
Compounds 21, 22, and 26 were obtained with higher yields than 20 and 25 for the presence of a carbonyl group in the ortho-position related to one of the hydroxyl groups on the phenyl ring. This fact can be elucidated due to the hydrogen bond between the hydroxyl group and the carbonyl group of the ethyl ester conferring a higher regiospecificity to the cyclization reaction, which is likely to occur at the position without hydrogen bonding. On the other hand, the stereospecificity during the intramolecular Diels-Alder reaction could be explained considering that in the transition state (21a), the methyl group adopted a coplanar structure in the chair configuration, so the exo-transition state is energetically more favorable than the endo-transition state, as shown in Scheme 3.

Scheme 3.
Reaction mechanism for the condensation between resorcinol derivative 15 and aldehyde 9a followed by the intramolecular Diels-Alder reaction.

Scheme 3.
Reaction mechanism for the condensation between resorcinol derivative 15 and aldehyde 9a followed by the intramolecular Diels-Alder reaction.
Garg and coworkers [30] established a method to obtain the (9R-)HHC diastereomer as a major product via the hydrogen-atom transfer reduction of D8THC, avoiding potentially risky catalytic hydrogenation conditions and poisonous heavy metals such as platinum or palladium. (9R-)HHC has been evaluated for the treatment of colon cancer [31] and ocular hypotony [32] with promising results. Furthermore, separately investigating the biological properties of the 9R-HHC (1) diastereomer would offer a comprehension of its pharmaceutical activity.
They employed tris(acetylacetonato)iron(III) that is an effective hydrogen atom donor catalyst for the radical reduction reactions in combination with thiophenol and silylbenzene to reduce d8THC (35b). Under these conditions, the mixture of diastereomers afforded 77% of yield and a ratio of 11:1 (9R-HHC:9S-HHC) (Scheme 6). It is outstanding that the hydrogen-atom transfer conditions furnish the highest diastereoselectivity in Scheme 5. Synthetic pathway using lewis acid, bases, organometallic reagents, and hydrogenation to obtain 9R-HHC-1a-c and 9S-HHC-6a-c.
Garg and coworkers [30] established a method to obtain the (9R-)HHC diastereomer as a major product via the hydrogen-atom transfer reduction of D8THC, avoiding potentially risky catalytic hydrogenation conditions and poisonous heavy metals such as platinum or palladium. (9R-)HHC has been evaluated for the treatment of colon cancer [31] and ocular hypotony [32] with promising results. Furthermore, separately investigating the biological properties of the 9R-HHC (1) diastereomer would offer a comprehension of its pharmaceutical activity.
They employed tris(acetylacetonato)iron(III) that is an effective hydrogen atom donor catalyst for the radical reduction reactions in combination with thiophenol and silylbenzene to reduce d8THC (35b). Under these conditions, the mixture of diastereomers afforded 77% of yield and a ratio of 11:1 (9R-HHC:9S-HHC) (Scheme 6). It is outstanding that the hydrogen-atom transfer conditions furnish the highest diastereoselectivity in favor of the equatorial orientation of the methyl group at position 9, demonstrating that 9R-HHC is energetically favorable.
as a major product via the hydrogen-atom transfer reduction of D8THC, avoidi potentially risky catalytic hydrogenation conditions and poisonous heavy metals such platinum or palladium. (9R-)HHC has been evaluated for the treatment of colon canc [31] and ocular hypotony [32] with promising results. Furthermore, separate investigating the biological properties of the 9R-HHC (1) diastereomer would offer comprehension of its pharmaceutical activity.
They employed tris(acetylacetonato)iron(III) that is an effective hydrogen ato donor catalyst for the radical reduction reactions in combination with thiophenol an silylbenzene to reduce d8THC (35b). Under these conditions, the mixture of diastereome afforded 77% of yield and a ratio of 11:1 (9R-HHC:9S-HHC) (Scheme 6). It is outstandi that the hydrogen-atom transfer conditions furnish the highest diastereoselectivity favor of the equatorial orientation of the methyl group at position 9, demonstrating th 9R-HHC is energetically favorable. Scheme 6. Synthesis of HHC diastereomers (1 and 7) via hydrogen-atom transfer reduction of D8THC (35b).
The key point in the synthetic route was a highly regio-and sereoselective S N 2 reaction to afford the 5-methyl-2-((prop-1-en-2-yl)cyclohexyl)benzene-1,3-diol scaffold (46) with the four stereocenters (C1, C2, C4, and C5) present in the final molecule ( Figure 3). The main disadvantage of this method is that 18 synthesis steps are required, entailing that the overall yield of (+)-machaeriol D is lower than 10%. Dethe [37] improved this procedure by applying an atom economical and protecting group-free synthetic strategy with less than six operational steps starting with R-(+) and S-(−)-limonene (47). This pathway provides the synthesis of both natural product 43 and its enantiomer 45 (Scheme 7).
The first step consists of the diastereoselective-coupling reaction between allylic alcohol 45 obtained from S-(−)limonene (47) with benzofuran-benzene-diol (51) in the presence of BF 3 ·OEt 2 followed by isomerization of the double bond to generate a 90% isolate yield of transhexahydrodibenzopyran compound 52. The high diastereoselectivity showed is due to the bulky isopropenyl group in the allyl alcohol. The second step involved the Prilezhaev epoxidation, which was carried out using 3-chlorobenzoperoxoic acid (m-CPBA) to afford a 74% yield of epoxide 53. The reaction was highly stereospecific, and it occurred from the α-face to obtain only one diastereoisomer. Interestingly, the regioselective opening of epoxide 53 occurs in the presence of the mixture of sodium cyanoborohydride (NaBH 3 CN) and BF 3 ·OEt 2 (1:1) to obtain the epimer of machaeriol-D (54) with a 45% overall yield. On the other hand, epoxide 53 undergoes a semipinacol rearrangement catalyzed by BF 3 ·OEt 2 to produce, regioselectively, ketone 55 with 82% of yield. The last step represents the reduction of 55 using sodium borohydride to afford the natural product (+)-machaeriol-D (43) in a 96% yield and 48% of overall yield.
Molecules 2023, 28, x FOR PEER REVIEW 16 of 59 2 h before death by stunning and decapitation [41]. Also, it was the major metabolite formed using the incubation of 9R-HHC with hepatic microsomes of rats, guinea pigs, and rabbits [42]. Interestingly, this compound was established to be closely seventeen times more active than Δ9 THC; for these reasons, great interest arose to develop the synthesis of it to deeply study its pharmacological activity in vitro and in vivo as well as its toxicity [43].
In the second step, 3-carbaldehyde (75) was treated with hydrogen under Pd/C to yield saturated carbaldehyde (76) as a racemic mixture (Scheme 11). Appayee [45] used DBU in MeOH followed by the in situ reduction of the epimerized product to achieve a good diastereoselectivity (5:1, d.r.) of cyclohexyl methanol (77) with a 60% yield after two steps. The conversion of 77 to t 2-((1R,2R,4R)-2-(2,6-dimethoxy-4-pentylphenyl)-4-(hydroxymethyl) cyclohexyl)propan-2-ol (80) was accomplished in four steps starting with the benzyl protection of the carbinol, then the direct oxidation of the silyl ethers' ether using the Jones reagent followed by treatment with trimethylsilyidiazomethanhexane resulting in cyclohexyl acetate (79) 16. Finally, the addition of methyllithium to compound 79 afforded cyclohexylpropan-2-ol (80) with an 85% yield after four steps (Scheme 11). The last step was a challenge due to the presence of a free tertiary alcohol group in 80 that triggers multiple dehydrated and rearranged products during the deprotection and cyclization reactions. For this reason, Appayee [45] decided to transform compound 80 into a terminal alkene and treated it with boron tribromide to obtain 9R-11hydroxyhexahydrocannabinol (71) with a 24% overall yield (Scheme 11). Scheme 11. Total synthesis of 9R-11-hydroxyhexahydrocannabinol developed by Appayee [45].
In this inverse-electron-demand Diels-Alder (IEDDA) reaction, an electron-rich dienophile (74) undergoes a 1,4 addition with an electron-poor diene (73). A tentative mechanism for this IEDDA was proposed by Appayee [46]. One equivalent of (S)-pyrrolidine-2carboxylic acid reacts with diene 73 to afford an enamine intermediary A, and the second equivalent of (S)-pyrrolidine-2-carboxylic acid reacts with dienophile (74) to furnish an iminium intermediary B. A and B go through a possible transition state, TS, to generate adduct C. The enolization of C leads to the formation of an enamine intermediary D. The last two steps of the mechanism comprise the elimination of the catalyst to give iminium intermediate E and the hydrolysis of E to furnish compound 75 (Scheme 12).

C-9 Ketocannabinoids: Different Enantioselective Synthetic Routes
The first synthesis of a C9-ketocannabinoid related to enantioenriched nabilone (88) was first developed by Archer and coworkers at Eli Lilly Company in 1977 [47]. Nabilone's structure is comparable to that of THC. Both compounds are a dibenzopyran core, with a dimethyl at C6, and a hydroxyl at C1. Contrasting THC, nabilone has a dimethylheptyl lipophilic chain at C3, a saturated ring at the terpene scaffold, and a ketone group instead of a methyl group in C9. Pertwee [48] demonstrated that due to these structural differences, nabilone is more potent than THC, producing higher cAMP agonist and Scheme 12. Mechanism for the IEDDA reaction proposed by Appayee [46]. The last step was a challenge due to the presence of a free tertiary alcohol group in 80 that triggers multiple dehydrated and rearranged products during the deprotection and cyclization reactions. For this reason, Appayee [45] decided to transform compound 80 into a terminal alkene and treated it with boron tribromide to obtain 9R-11hydroxyhexahydrocannabinol (71) with a 24% overall yield (Scheme 11).

C-9 Ketocannabinoids: Different Enantioselective Synthetic Routes
The first synthesis of a C9-ketocannabinoid related to enantioenriched nabilone (88) was first developed by Archer and coworkers at Eli Lilly Company in 1977 [47]. Nabilone's structure is comparable to that of THC. Both compounds are a dibenzopyran core, with a dimethyl at C6, and a hydroxyl at C1. Contrasting THC, nabilone has a dimethylheptyl lipophilic chain at C3, a saturated ring at the terpene scaffold, and a ketone group instead of a methyl group in C9. Pertwee [48] demonstrated that due to these structural differences, nabilone is more potent than THC, producing higher cAMP agonist and [ 35 S]GTPγS binding affinity in mouse brain tissue. In 1975, Lemberger and Rowe [49] reported the use of nabilone administration in humans, pointing out that the behavioral effects begin at about 1 h after administration, and last for 8-9 h. In total, 5 mg of nabilone produced dry mouth, euphoria, tachycardia, and postural hypotension. These effects were insignificant at 2.5 mg, and lacking at 1 mg. Later, clinical studies advocated that nabilone may be effective in relieving agitation in patients with dementia [50,51], nightmares in patients with posttraumatic stress disorder [52], and non-motor symptoms in patients with Parkinson's disease [53]. This motivated many research groups to improve the synthetic procedure proposed by the Eli Lilly Company and try to obtain pure diastereomers instead of the racemic mixture.

Cannabinoid Lactones Modified in the C-Ring
Makriyannis [58] substituted the C-ring in the nabilone structure with a sevenmembered lactone. The goal of this work was to incorporate a labile group as lactone into the C-9 ketocannabinoid lead scaffold to improve pharmacokinetic/pharmacodynamic properties of cannabinoids and mimic their cannabinergic effects.

Cannabinoid Lactones Modified in the C-Ring
Makriyannis [58] substituted the C-ring in the nabilone structure with a seven-membered lactone. The goal of this work was to incorporate a labile group as lactone into the C-9 ketocannabinoid lead scaffold to improve pharmacokinetic/pharmacodynamic properties of cannabinoids and mimic their cannabinergic effects.
The Baeyer-Villiger oxidation rearrangement is a key point in this synthetic pathway; hence, we decided to include the reaction mechanism. It involves the formation of a seven-member cyclic ortho-ester from a six-member cyclic ketone using peroxyacids as an oxidant. The reaction implies the initial addition of peroxide to the carbonyl carbon to obtain an 88a adduct, which undergoes a rearrangement to obtain the intermediate α−acylperoxy hemiacetals' (Criegee) intermediary 96. The last step compromises the alkyl migration to give the two regioisomers 97a and 97b with a ratio of 2.7:1 (97a:97b) (Scheme 15A). The group anti-periplanar alignment to the dissociating peroxide bond is expected to have a higher migratory ability for conformational and stereoelectronic requirements with the lower energy in the dipole interaction. Thus, the formation of lactone 97a is favored over regioisomer 97b. Mikami [59] investigated the regioselectivity of the Baeyer-Villiger reaction in six-membered cyclic ketones and developed a regioselective procedure to afford only one regioisomer of lactones using aqueous hydrogen peroxide as an oxidant and Sn-zeolite as a catalyst (Scheme 15B). It could be applied to the synthesis of (5aR,11bR)-11-hydroxy-6,6-dimethyl-9-(2-methyloctan-2-yl)-4,5,5a,6tetrahydro-1H-oxepino[4,3-c]chromen-3(11bH)-one (99a).

Hydrogenated Bicyclic Cannabidiol Analogs
Cannabidiol (CBD, 34b) is a naturally occurring compound biosynthesized within the Cannabis sativa plant. CBD has gained significant attention in recent years due to its potential therapeutic effects in treating multiple ailments while exhibiting minimal to no psychoactive properties. CBD has been reported to exhibit various effects on the human body. Studies suggest that it possesses anti-inflammatory, analgesic (pain-relieving), anxiolytic (anti-anxiety), and neuroprotective properties [60]. CBD has also shown potential in the treatment of epilepsy, schizophrenia, and other psychiatric disorders [61]. Additionally, it may have antioxidant and anticancer properties, through studies hypothesizing the mechanisms that CBD might enact on [62].
The mechanisms through which CBD exerts its effects are complex and multifaceted. CBD interacts with several molecular targets in the body, including cannabinoid receptors (CB1 and CB2), serotonin receptors (5-HT1A), and transient receptor potential (TRP) channels [63]. However, CBD does not bind strongly to these receptors, and its effects are believed to be largely mediated through the indirect modulation of endogenous neurotransmitter systems. CBD's interaction with the endocannabinoid system (ECS) is of particular importance. Although CBD has low affinity for cannabinoid receptors, it can influence the ECS by inhibiting the enzyme fatty acid amide hydrolase (FAAH), which is responsible for the breakdown of anandamide, an endogenous cannabinoid. By inhibiting FAAH, CBD increases anandamide levels, leading to potential therapeutic effects [64]. Furthermore, CBD has been found to modulate various signaling pathways and molecular targets involved in inflammation, oxidative stress, and neurotransmission. It affects the release and uptake of neurotransmitters such as serotonin, dopamine, and glutamate, contributing to its anxiolytic and antipsychotic properties [65].
Considering the therapeutic applications of CBD and its low toxicity, a marked interest has emerged in the design of new analogs of hydrogenated bicyclic CBD and quinones [66] to study its pharmacological and clinical effects.
Molecules 2023, 28, x FOR PEER REVIEW 24 of 59 the cyclohexane/terpene ring. This difference in 3D shapes strongly suggests that one of the isomers could be far more active, interacting with the binding targets with increased affinity and specificity.

Machaeridiols and Their Synthetic Analogs
Natural machaeridiol compounds have the skeleton configuration at 1R and 2R positions opposite to those at 1S and 2S positions of dihydro-CBD and the same as the enantiomer 5S position. Also, the machaeridiol chemotype is like dihydro-CBD, with the n-alkyl moiety replaced by steryl and benzofuranyl forms. HHDBP-type phytocannabinoids displayed potent activity against Staphylococcus aureus (vancomycinresistant), Enterococcus faecium, and E. faecalis such as machaeridiols A [67,68]. The biological activities and interesting structural design of this class of natural compounds have inspired synthetic efforts directed toward their total syntheses.

Machaeridiols and Their Synthetic Analogs
Natural machaeridiol compounds have the skeleton configuration at 1R and 2R positions opposite to those at 1S and 2S positions of dihydro-CBD and the same as the enantiomer 5S position. Also, the machaeridiol chemotype is like dihydro-CBD, with the n-alkyl moiety replaced by steryl and benzofuranyl forms. HHDBP-type phytocannabinoids displayed potent activity against Staphylococcus aureus (vancomycin-resistant), Enterococcus faecium, and E. faecalis such as machaeridiols A [67,68]. The biological activities and interesting structural design of this class of natural compounds have inspired synthetic efforts directed toward their total syntheses.
Williamson [70] and later Sarlah [71] developed different ways to synthesize CBE, 121 starting with CBD (34b). On the first route, CBD was completely silylated using N,O bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by chemoselective epoxidation to obtain 1R,2R,3R,6S-silyl epoxide (125), which was deprotected in the presence of sodium hydroxide in methanol to achieve CBE (121) in a 52% yield (Scheme 19). In the second way, the CBD underwent full acetylation and then chemoselective oxidation to give 1R,2R,3R,6S-acetyl epoxide (128). Epoxide 128 was exposed to an excess of potassium carbonate in methanol to deliver CBE (121) with a 42% overall yield. Williamson [70] carried out the epoxidation without protecting the CBD, which reversed the major facial selectivity of the epoxidation to obtain 1S,2R,3R,6R-epoxide (126) in an 83% yield. However, the cyclization of epoxide 126 to generate CBE failed (Scheme 19). It is due to a higher energy barrier for the equatorial attack of bases on cyclohexane-derived epoxides [72].
Williamson [70] and later Sarlah [71] developed different ways to synthesize CBE, 121 starting with CBD (34b). On the first route, CBD was completely silylated using N,O bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by chemoselective epoxidation to obtain 1R,2R,3R,6S-silyl epoxide (125), which was deprotected in the presence of sodium hydroxide in methanol to achieve CBE (121) in a 52% yield (Scheme 19). In the second way, the CBD underwent full acetylation and then chemoselective oxidation to give 1R,2R,3R,6Sacetyl epoxide (128). Epoxide 128 was exposed to an excess of potassium carbonate in methanol to deliver CBE (121) with a 42% overall yield. Williamson [70] carried out the epoxidation without protecting the CBD, which reversed the major facial selectivity of the epoxidation to obtain 1S,2R,3R,6R-epoxide (126) in an 83% yield. However, the cyclization of epoxide 126 to generate CBE failed (Scheme 19). It is due to a higher energy barrier for the equatorial attack of bases on cyclohexane-derived epoxides [72].

Saturated Quinonoid Cannabinoid
There are various other saturated cannabinoids that have been explored and studied. Some of which include a variety of quinol compounds. Quinones have various biological activities and several natural and synthetic quinone compounds are currently used as therapeutic drugs. One particular cannabinoid quinone (HU-331: was synthesized in 1968 to address the question of cannabinoids giving a purple color Scheme 20. Synthetic pathway proposed by Sarlah [71] and Echavarren [73] to obtain the natural products ACBM (122) and R-CBM (123) and the synthetic diastereomer S-CBM (133).

Scheme 21.
Oxidation of H2-CBD (101a) and H4-CBD (103a) to obtain their corresponding quinone derivatives in the presence of oxygen.

Scheme 21.
Oxidation of H 2 -CBD (101a) and H 4 -CBD (103a) to obtain their corresponding quinone derivatives in the presence of oxygen.

Biological Studies of Saturated Cannabinoids
Although saturated cannabinoids have been known for about 100 years, no absorption, distribution, metabolism, and excretion (ADME) studies have been published in peerreview journals. It is important to consider that HHC has invaded the recreational market in the last 2 years and its consumption by inhaling, ingesting in the form of edibles, or taking it sublingually with oils could trigger psychotropic effects by not knowing the proper dosages and side effects of this product and its analogs.
For this reason, research on the mechanism of action, the interaction in the human organism, and the new biological applications of HHCs and their analogs should be a priority in research projects.
In this section of the review, we compiled all the data on the affinities of saturated cannabinoids for CB1 and CB2 receptors and their relationship with the different functionalities in the HHC scaffold, considering the five distinct regions (terpene moiety, ring B, resorcinol core, lipid tail, and stereocenters) or the four main pharmacophores (alkyl side chain, phenolic hydroxyl group, northern aliphatic group, and southern substituent in the pyran ring) in the HHC structure, which are important for cannabimimetic receptor affinities ( Figure 5).

Biological Studies of Saturated Cannabinoids
Although saturated cannabinoids have been known for about 100 years, no absorption, distribution, metabolism, and excretion (ADME) studies have been published in peer-review journals. It is important to consider that HHC has invaded the recreational market in the last 2 years and its consumption by inhaling, ingesting in the form of edibles, or taking it sublingually with oils could trigger psychotropic effects by not knowing the proper dosages and side effects of this product and its analogs.
For this reason, research on the mechanism of action, the interaction in the human organism, and the new biological applications of HHCs and their analogs should be a priority in research projects.
In this section of the review, we compiled all the data on the affinities of saturated cannabinoids for CB1 and CB2 receptors and their relationship with the different functionalities in the HHC scaffold, considering the five distinct regions (terpene moiety, ring B, resorcinol core, lipid tail, and stereocenters) or the four main pharmacophores (alkyl side chain, phenolic hydroxyl group, northern aliphatic group, and southern substituent in the pyran ring) in the HHC structure, which are important for cannabimimetic receptor affinities ( Figure 5). The modification in the terpene moiety determines the role of the ring rigidity and whether the introduction of hydrogen bond donors and acceptors could influence the affinity and selectivity for both CB1 and CB2 receptors. The alteration of the resorcinol The modification in the terpene moiety determines the role of the ring rigidity and whether the introduction of hydrogen bond donors and acceptors could influence the affinity and selectivity for both CB1 and CB2 receptors. The alteration of the resorcinol ring allows for examining the effect of the free hydroxyl group, protecting forming ethers, oxidizing forming quinones, or removed on the biological activity of hydrogenated cannabinoids. The alkyl chain and stereocenters permit to an evaluation of how geometric constraints and lipophilicity influence binding pockets. Finally, it is important to determine the difference between bicyclic cannabinoids (CBD analogs, ring B opened) and tricyclic cannabinoids (THC analogs, ring B closed) in receptor affinity.
The search to comprehend the molecular basis of the pharmacological effects of cannabinoids led to the identification and characterization of CB receptors. The cannabinoid receptors are membrane-bound receptors that belong to a superfamily of G-protein coupled receptors (GPCRs). To date, two CB receptors, CB1 and CB2, have been isolated, cloned, and expressed. The first cannabinoid receptor (CB1) was discovered when Matsuda cloned and expressed this GPCR from rat brains in 1990 [99] followed by the expression of human CB1 in 1991 by Gerard [100]. In 1993, Munro found, cloned, and expressed a second cannabinoid receptor (CB2) within the preparation of a human promyelocytic leukemia cell line (HL60) [101].
Macheriols and machaeridiols are important types of hexahydrodibenzopyrancannabinoids. Macheriols are characterized by having a chromane core and an ABC tricyclic system, structurally similar to HHC, and machaeridiols are defined by the open B pyran ring, which resembles H 4 CBD [102]. The main difference lies in the inversion of stereocenters on position 6a and 10a for machaeriol or 1 and 2 for machaeridiols. Also, these compounds showed an aralkyl group as a side chain instead of a lipophilic chain as HHC and H 4 CBD.
Thapa et al. [89] demonstrated that anticancer effects of novel machaeridiol and machaeriol analogs imply the inhibition of cell proliferation and tumor angiogenesis and recently, Muhammad et al. [102] examined the in vitro cytotoxicity of some natural macheriols and machaeridiols against human solid tumor cell lines such as SK-MEL, KB, BT-549, SK-OV-3, and HeLa. They confirmed that the combination (1:1) of compound 39 (macheriol B) and compound 167 (machaeridiol B) exhibited activity against the five human cancer cell lines with an IC 50 between 26 and 33 µg/mL. Table 3 reveals that machaeridiols A, B, and C (106, 167, and 168) show selective binding affinities for CB2 receptors; however, machaeriol C and D (39 and 43) exhibit affinities for both CB1 and CB2 receptors.
Chittiboyina et al. [103] designed a synthetic machaeriol (compound 166, Table 3) that is a CB2-selective agonist, which is characterized by a benzothiophene moiety in the side chain. They performed in silico molecular docking experiments to explain the binding affinities of compound 166 into the active sites of CB1 and CB2 receptors' protein crystal structures using Maestro, Schrödinger ( Figure 6A). This compound showed π-π stacking interactions between hexahydrochromane and benzothiophene cores with the residues Phe170, Phe268, and Trp279 of the CB1 receptor. In addition, 166 generated hydrophobic interactions with a series of aquaphobic residues involving Phe108, Phe174, Phe177, Leu193, Val196, Phe200, Ile267, Trp279, Trp356, Leu359, Phe379, Ala380, and Cys386. In a similar fashion, compound 166 exhibited π-π stacking and hydrophobic interactions with CB2 residues. However, the major difference lay in the H-bonding shown between the hydroxyl group of the resorcinol ring and Ser285 ( Figure 6B, marked with a purple circle), which is an essential residue for CB2 receptor activity. (2-methyloctan-2-yl)benzene-1,3-diol (103b, Table 2) have affinity for the cannabinoid CB1 receptor. It means that by removing the double bond from ring C and from the southern aliphatic chain, the ability to bind to the CB1 receptor increases. Also, by branching the lipophilic chain incorporating two methyl groups, the affinity for the CB1 receptor (comparing compounds 103a and 103b) was improved. Ben-Shabat [9] demonstrated that the anti-inflammatory capacity of these compounds owes its origin to the effect on the production of reactive oxygen intermediates (ROIs), nitric oxide (NO), and tumor necrosis factor (TNF). Moreover, Ben-Shabat [9] concluded that the activation of such mediators is not directly through central cannabinoid receptor CB1 because compound 103b showed decreased suppressive effects on ROI, NO, and TNF-R production compared to compound 103a (Table 2). (2-methyloctan-2-yl)benzene-1,3-diol (103b, Table 2) have affinity for the cannabinoid CB1 receptor. It means that by removing the double bond from ring C and from the southern aliphatic chain, the ability to bind to the CB1 receptor increases. Also, by branching the lipophilic chain incorporating two methyl groups, the affinity for the CB1 receptor (comparing compounds 103a and 103b) was improved. Ben-Shabat [9] demonstrated that the anti-inflammatory capacity of these compounds owes its origin to the effect on the production of reactive oxygen intermediates (ROIs), nitric oxide (NO), and tumor necrosis factor (TNF). Moreover, Ben-Shabat [9] concluded that the activation of such mediators is not directly through central cannabinoid receptor CB1 because compound 103b showed decreased suppressive effects on ROI, NO, and TNF-R production compared to compound 103a (Table 2). pentylbenzene-1,3-diol (103a, Table 2) and 2-((1S,2S)-2-isopropyl-5-methylcyclohexyl)-5-(2-methyloctan-2-yl)benzene-1,3-diol (103b, Table 2) have affinity for the cannabinoid CB1 receptor. It means that by removing the double bond from ring C and from the southern aliphatic chain, the ability to bind to the CB1 receptor increases. Also, by branching the lipophilic chain incorporating two methyl groups, the affinity for the CB1 receptor (comparing compounds 103a and 103b) was improved. Ben-Shabat [9] demonstrated that the anti-inflammatory capacity of these compounds owes its origin to the effect on the production of reactive oxygen intermediates (ROIs), nitric oxide (NO), and tumor necrosis factor (TNF). Moreover, Ben-Shabat [9] concluded that the activation of such mediators is not directly through central cannabinoid receptor CB1 because compound 103b showed decreased suppressive effects on ROI, NO, and TNF-R production compared to compound 103a (Table 2). Macheriols and machaeridiols are important types of hexahydrodibenzopyrancannabinoids. Macheriols are characterized by having a chromane core and an ABC tricyclic system, structurally similar to HHC, and machaeridiols are defined by the open B pyran ring, which resembles H4CBD [102]. The main difference lies in the inversion of stereocenters on position 6a and 10a for machaeriol or 1 and 2 for machaeridiols. Also, these compounds showed an aralkyl group as a side chain instead of a lipophilic chain as HHC and H4CBD.
Thapa et al. [89] demonstrated that anticancer effects of novel machaeridiol and machaeriol analogs imply the inhibition of cell proliferation and tumor angiogenesis and recently, Muhammad et al. [102] examined the in vitro cytotoxicity of some natural macheriols and machaeridiols against human solid tumor cell lines such as SK-MEL, KB, BT-549, SK-OV-3, and HeLa. They confirmed that the combination (1:1) of compound 39 (macheriol B) and compound 167 (machaeridiol B) exhibited activity against the five human cancer cell lines with an IC50 between 26 and 33 µg/mL. Table 3 reveals that machaeridiols A, B, and C (106, 167, and 168) show selective binding affinities for CB2 receptors; however, machaeriol C and D (39 and 43) exhibit affinities for both CB1 and CB2 receptors. Macheriols and machaeridiols are important types of hexahydrodibenzopyrancannabinoids. Macheriols are characterized by having a chromane core and an ABC tricyclic system, structurally similar to HHC, and machaeridiols are defined by the open B pyran ring, which resembles H4CBD [102]. The main difference lies in the inversion of stereocenters on position 6a and 10a for machaeriol or 1 and 2 for machaeridiols. Also, these compounds showed an aralkyl group as a side chain instead of a lipophilic chain as HHC and H4CBD.
Thapa et al. [89] demonstrated that anticancer effects of novel machaeridiol and machaeriol analogs imply the inhibition of cell proliferation and tumor angiogenesis and recently, Muhammad et al. [102] examined the in vitro cytotoxicity of some natural macheriols and machaeridiols against human solid tumor cell lines such as SK-MEL, KB, BT-549, SK-OV-3, and HeLa. They confirmed that the combination (1:1) of compound 39 (macheriol B) and compound 167 (machaeridiol B) exhibited activity against the five human cancer cell lines with an IC50 between 26 and 33 µg/mL. Table 3 reveals that machaeridiols A, B, and C (106, 167, and 168) show selective binding affinities for CB2 receptors; however, machaeriol C and D (39 and 43) exhibit affinities for both CB1 and CB2 receptors.  Macheriols and machaeridiols are important types of hexahydrodibenzopyrancannabinoids. Macheriols are characterized by having a chromane core and an ABC tricyclic system, structurally similar to HHC, and machaeridiols are defined by the open B pyran ring, which resembles H4CBD [102]. The main difference lies in the inversion of stereocenters on position 6a and 10a for machaeriol or 1 and 2 for machaeridiols. Also, these compounds showed an aralkyl group as a side chain instead of a lipophilic chain as HHC and H4CBD.
Thapa et al. [89] demonstrated that anticancer effects of novel machaeridiol and machaeriol analogs imply the inhibition of cell proliferation and tumor angiogenesis and recently, Muhammad et al. [102] examined the in vitro cytotoxicity of some natural macheriols and machaeridiols against human solid tumor cell lines such as SK-MEL, KB, BT-549, SK-OV-3, and HeLa. They confirmed that the combination (1:1) of compound 39 (macheriol B) and compound 167 (machaeridiol B) exhibited activity against the five human cancer cell lines with an IC50 between 26 and 33 µg/mL. Table 3 reveals that machaeridiols A, B, and C (106, 167, and 168) show selective binding affinities for CB2 receptors; however, machaeriol C and D (39 and 43) exhibit affinities for both CB1 and CB2 receptors.    Chittiboyina et al. [103] designed a synthetic machaeriol (compound 166, Table 3) that is a CB2-selective agonist, which is characterized by a benzothiophene moiety in the side chain. They performed in silico molecular docking experiments to explain the binding affinities of compound 166 into the active sites of CB1 and CB2 receptors' protein crystal structures using Maestro, Schrödinger ( Figure 6A). This compound showed π-π stacking interactions between hexahydrochromane and benzothiophene cores with the residues Phe170, Phe268, and Trp279 of the CB1 receptor. In addition, 166 generated hydrophobic interactions with a series of aquaphobic residues involving Phe108, Phe174, Phe177, Leu193, Val196, Phe200, Ile267, Trp279, Trp356, Leu359, Phe379, Ala380, and Cys386. In a similar fashion, compound 166 exhibited π-π stacking and hydrophobic interactions with  Chittiboyina et al. [103] designed a synthetic machaeriol (compound 166, Table 3) that is a CB2-selective agonist, which is characterized by a benzothiophene moiety in the side chain. They performed in silico molecular docking experiments to explain the binding affinities of compound 166 into the active sites of CB1 and CB2 receptors' protein crystal structures using Maestro, Schrödinger ( Figure 6A). This compound showed π-π stacking interactions between hexahydrochromane and benzothiophene cores with the residues Phe170, Phe268, and Trp279 of the CB1 receptor. In addition, 166 generated hydrophobic interactions with a series of aquaphobic residues involving Phe108, Phe174, Phe177, Leu193, Val196, Phe200, Ile267, Trp279, Trp356, Leu359, Phe379, Ala380, and Cys386. In a similar fashion, compound 166 exhibited π-π stacking and hydrophobic interactions with  [103] 168 (Machaeridiol C) >1000 -1.11 --CB2 selective agonists [103] Chittiboyina et al. [103] designed a synthetic machaeriol (compound 166, Table 3) that is a CB2-selective agonist, which is characterized by a benzothiophene moiety in the side chain. They performed in silico molecular docking experiments to explain the binding affinities of compound 166 into the active sites of CB1 and CB2 receptors' protein crystal structures using Maestro, Schrödinger ( Figure 6A). This compound showed π-π stacking interactions between hexahydrochromane and benzothiophene cores with the residues Phe170, Phe268, and Trp279 of the CB1 receptor. In addition, 166 generated hydrophobic interactions with a series of aquaphobic residues involving Phe108, Phe174, Phe177, Leu193, Val196, Phe200, Ile267, Trp279, Trp356, Leu359, Phe379, Ala380, [103] 168 (Machaeridiol C) >1000 -1.11 --CB2 selective agonists [103] Chittiboyina et al. [103] designed a synthetic machaeriol (compound 166, Table 3) that is a CB2-selective agonist, which is characterized by a benzothiophene moiety in the side chain. They performed in silico molecular docking experiments to explain the binding affinities of compound 166 into the active sites of CB1 and CB2 receptors' protein crystal structures using Maestro, Schrödinger ( Figure 6A). This compound showed π-π stacking interactions between hexahydrochromane and benzothiophene cores with the residues Phe170, Phe268, and Trp279 of the CB1 receptor. In addition, 166 generated hydrophobic interactions with a series of aquaphobic residues involving Phe108, Phe174, Phe177, Leu193, Val196, Phe200, Ile267, Trp279, Trp356, Leu359, Phe379, Ala380, and Cys386. In a We consider it essential to carry out a more in-depth study of SAR on machaeriol and machaeridiol derivatives to achieve novel analogs with better CB2 receptor selectivity, focusing on the side chain and the stereocenters of the HHDBP scaffold (46). Tables 4-6 show how the four main pharmacophores influence the binding affinities of nonclassical and hybrid saturated tricyclic cannabinoids for CB1 and CB2 receptors in in vitro experiments and SAR studies.

Southern Aliphatic Hydroxyl Chain (SAH)
Modification of SAH generate a family of non-classical cannabinoids that have not been found in the cannabis plant [56,104,105]. First, we focus on the effect of the orientation of the SAH group. For this, Makriyannis [105] synthesized compounds 197 and 198, demonstrating that the epimer (6S,6aR,9R,10aS)-6-(2-hydroxyethyl)-6-methyl-3pentyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-1, 9-diol (197), with the hydroxyethyl group being in the equatorial position, has greater affinity for both receptors CB1 and CB2, resulting in more favorable ligand-receptor interaction (Table 4). Second, Makriyannis carried out SAR studies to examine the role of the hydroxyalkyl chain length and bulk in the activity of this scaffold. The binding affinities of compounds 188, 189, and 190 indicate little change in the CB1 and CB2 receptor affinity with increasing chain length. From the receptor binding data that display compounds 186, 191, and 190, it can be concluded that the conformation of the side chain is not important for ligand-receptor interaction since the alkyne (191) and alkene (186) analogs exhibit similar receptor affinity to that of the hydroxyalkyl analog (190). When incorporating a halogen such as iodine (compound, 199), the binding affinity for the CB1 and CB2 receptor decreases. From these results, it can be concluded that while the relative configuration 6-axial or 6-equatorial of the SAH appears to be critical, the length and the conformation of the southern hydroxyl chain are of lesser effect in determining the cannabinoid activity. Including a halogen atom is reflected in the loss of affinity for CB1 and CB2 receptors. We consider it essential to carry out a more in-depth study of SAR on machaeriol and machaeridiol derivatives to achieve novel analogs with better CB2 receptor selectivity, focusing on the side chain and the stereocenters of the HHDBP scaffold (46). Tables 4-6 show how the four main pharmacophores influence the binding affinities of nonclassical and hybrid saturated tricyclic cannabinoids for CB1 and CB2 receptors in in vitro experiments and SAR studies.

Southern Aliphatic Hydroxyl Chain (SAH)
Modification of SAH generate a family of non-classical cannabinoids that have not been found in the cannabis plant [56,104,105]. First, we focus on the effect of the orientation of the SAH group. For this, Makriyannis [105] synthesized compounds 197 and 198, demonstrating that the epimer (6S,6aR,9R,10aS)-6-(2-hydroxyethyl)-6-methyl-3-pentyl-6a,7,8,9,10,10ahexahydro-6H-benzo[c]chromene-1, 9-diol (197), with the hydroxyethyl group being in the equatorial position, has greater affinity for both receptors CB1 and CB2, resulting in more favorable ligand-receptor interaction (Table 4). Second, Makriyannis carried out SAR studies to examine the role of the hydroxyalkyl chain length and bulk in the activity of this scaffold. The binding affinities of compounds 188, 189, and 190 indicate little change in the CB1 and CB2 receptor affinity with increasing chain length. From the receptor binding data that display compounds 186, 191, and 190, it can be concluded that the conformation of the side chain is not important for ligand-receptor interaction since the alkyne (191) and alkene (186) analogs exhibit similar receptor affinity to that of the hydroxyalkyl analog (190). When incorporating a halogen such as iodine (compound, 199), the binding affinity for the CB1 and CB2 receptor decreases. From these results, it can be concluded that while the relative configuration 6-axial or 6-equatorial of the SAH appears to be critical, the length and the conformation of the southern hydroxyl chain are of lesser effect in determining the cannabinoid activity. Including a halogen atom is reflected in the loss of affinity for CB1 and CB2 receptors.

Northern Aliphatic Group (NAG)
Regarding NAG, we examined the role of the stereochemistry at C-10, the length of the C-10 substituent, and the functionality at C-10 in the cannabimimetic activity. The binding affinities' data for CB1 and CB2 receptors appear in Tables 4-6. Table 6, which represents novel hydrogenated adamantyl cannabinoids, shows that all 10β-epimers (the equatorial orientation of the C-10-alkyl chain) improve CB1 and CB2 affinities compared to the 9α-epimers. The length of the C-10-alkyl chain does not affect the CB1 and CB2 affinities comparing compounds 219 and 224 in Table 6. The iodo-methyl derivative (221) sharply decreased CB1/CB2 affinities, revealing poor steroelectronic interactions at CB1 and CB2 residues. Judging by the data of binding affinities of pair compounds 217/224 (Table 6) and 93/89 (Table 4), the functionality on C-10 revealed a better CB1/CB2 affinity of CH 2 OH compared with OH. Judging by the data of binding affinities of pair compounds 217/224 ( Table 6) and 93/89 (Table 4), C-10 functionality (CH 2 OH) revealed better CB1/CB2 than the OH group. In general, a hydroxyl group at the northern section of the tricyclic cannabinoids boosts the ligand's affinity for both CB receptors. Contrasting the CB1/CB2 affinity value of compound 93, Table 4 (3.0/2.1) and 179, Table 4 (0.6/2.65), it proves that the introduction of the azido group (179) increases the affinity for the CB1 receptor and it remains the same (the affinity for the CB1 receptor).

Phenolic Group
Cannabinoid derivatives in which the hydroxyl group in the resorcinol core was removed or substituted by an alkyl chain to generate an ether group significantly decrease ligand binding to CB1, displaying better selectivity for the CB2 receptor (comparing compounds 200 and 201, Table 5). Compound 201, the corresponding methyl ether of 200, exhibits more than 2000-fold CB2 selectivity. Interestingly, affinity to CB2 is only faintly altered by these changes.

Alkyl Side Chain
The manipulation of the electronics and conformational flexibility of the lipophilic side chain reveals the complexity and specificity of the cannabinoid-binding pocket as Tables 4 and 5 show.
Ramification between C-1 and C-2 in the side chain specifically introducing a dimethyl or cyclopentyl group as shown in compounds 172/173, 184/185, and 181/185 leads to increased receptor affinity and selectivity, obtaining a CB1 receptor selective antagonist when it introduces a four-carbon cycle between C-1 and C-2 (compound 193). Regarding unsaturation at the lipidic chain, no further increase in potency is noted when C-2 and C-3 are joined by a double bond, as illustrated in compound 206 (alkene) compared with 207 (unsaturated chain) or compound 182, which has a double bond between C-1 and C-2 compared with 184 (alkane). However, in compound 181, which presents a triple bond at C-1 and C-2 , the CB2-biding affinity decreases relating to 182 (alkene) and 184 (alkane). The addition of a halogen group and the end of the side chain slightly affects the receptor affinity (compounds 193, 195, and 196). Targeted covalent inhibitors (TCIs) represent an interesting development in cannabinoid ligands. Two major types of covalently activated lipidic chains have been employed as TCIs, those upholding electrophilic or photoactivatable functionality . For example, compounds 170, 174, and 177, which have attached an azide (-N 3 , photoactivatable moiety), isothiocyanate (-NCS, electrophilic functional group), or cyano (-NC, electrophilic functional group) functionality, respectively, reduce the CB1 and CB2 receptor affinity (Table 4). Makriyannis [56] carried out molecular docking studies based on the hCB1 crystal structure (PDB: 5XR8). They explored the interactions of typical lipid-chain agonists with the CB1 receptor through molecular docking, revealing that all agonists adopt an L-shape configuration in the orthosteric-binding pocket. The interactions between the tricyclic HHC core system and CB1 are essentially hydrophobic and aromatic. For example, the π-π interactions with Phe268, Phe379, Phe189, and Phe177 residues and phenolic hydroxyl form a hydrogen bond with Ser383. π interactions with Phe268, Phe379, Phe189, and Phe177 residues and phenolic hydroxyl form a hydrogen bond with Ser383. π interactions with Phe268, Phe379, Phe189, and Phe177 residues and phenolic hydroxyl form a hydrogen bond with Ser383. π interactions with Phe268, Phe379, Phe189, and Phe177 residues and phenolic hydroxyl form a hydrogen bond with Ser383. π interactions with Phe268, Phe379, Phe189, and Phe177 residues and phenolic hydroxyl form a hydrogen bond with Ser383. π interactions with Phe268, Phe379, Phe189, and Phe177 residues and phenolic hydroxyl form a hydrogen bond with Ser383. π interactions with Phe268, Phe379, Phe189, and Phe177 residues and phenolic hydroxyl form a hydrogen bond with Ser383. π interactions with Phe268, Phe379, Phe189, and Phe177 residues and phenolic hydroxyl form a hydrogen bond with Ser383.

Seven-Membered Lactone and Quinone in the Terpene Region
Incorporating a seven-membered lactone in ring C of the HHC scaffold generates a selective rCB1 agonist compound (99a, Table 7). It is interesting that its regioisomer (99b) did not display selectivity for rCB1 receptors. This confirms that the spatial configuration of the diastereomers plays a crucial role in the interactions with CB1 and CB2 receptors. Based on these results, we would propose the study of the affinities of a six-membered cannabinoid lactone for cannabinoid receptors.

Seven-Membered Lactone and Quinone in the Terpene Region
Incorporating a seven-membered lactone in ring C of the HHC scaffold generates a selective rCB1 agonist compound (99a, Table 7). It is interesting that its regioisomer (99b) did not display selectivity for rCB1 receptors. This confirms that the spatial configuration of the diastereomers plays a crucial role in the interactions with CB1 and CB2 receptors. Based on these results, we would propose the study of the affinities of a six-membered cannabinoid lactone for cannabinoid receptors.

Seven-Membered Lactone and Quinone in the Terpene Region
Incorporating a seven-membered lactone in ring C of the HHC scaffold generates a selective rCB1 agonist compound (99a, Table 7). It is interesting that its regioisomer (99b) did not display selectivity for rCB1 receptors. This confirms that the spatial configuration of the diastereomers plays a crucial role in the interactions with CB1 and CB2 receptors. Based on these results, we would propose the study of the affinities of a six-membered cannabinoid lactone for cannabinoid receptors.

Seven-Membered Lactone and Quinone in the Terpene Region
Incorporating a seven-membered lactone in ring C of the HHC scaffold generates a selective rCB1 agonist compound (99a, Table 7). It is interesting that its regioisomer (99b) did not display selectivity for rCB1 receptors. This confirms that the spatial configuration of the diastereomers plays a crucial role in the interactions with CB1 and CB2 receptors. Based on these results, we would propose the study of the affinities of a six-membered cannabinoid lactone for cannabinoid receptors.
Cannabinoid-receptor binding affinities presented in Table 8 demonstrated that the introduction of the 1,4-quinone moiety in ring C (compounds 139 and 140) led to the loss of affinity towards cannabinoid receptors CB1 and CB2.

Seven-Membered Lactone and Quinone in the Terpene Region
Incorporating a seven-membered lactone in ring C of the HHC scaffold generates a selective rCB1 agonist compound (99a, Table 7). It is interesting that its regioisomer (99b) did not display selectivity for rCB1 receptors. This confirms that the spatial configuration of the diastereomers plays a crucial role in the interactions with CB1 and CB2 receptors. Based on these results, we would propose the study of the affinities of a six-membered cannabinoid lactone for cannabinoid receptors. Cannabinoid-receptor binding affinities presented in Table 8 demonstrated that the introduction of the 1,4-quinone moiety in ring C (compounds 139 and 140) led to the loss of affinity towards cannabinoid receptors CB1 and CB2.

Seven-Membered Lactone and Quinone in the Terpene Region
Incorporating a seven-membered lactone in ring C of the HHC scaffold generates a selective rCB1 agonist compound (99a, Table 7). It is interesting that its regioisomer (99b) did not display selectivity for rCB1 receptors. This confirms that the spatial configuration of the diastereomers plays a crucial role in the interactions with CB1 and CB2 receptors. Based on these results, we would propose the study of the affinities of a six-membered cannabinoid lactone for cannabinoid receptors. Cannabinoid-receptor binding affinities presented in Table 8 demonstrated that the introduction of the 1,4-quinone moiety in ring C (compounds 139 and 140) led to the loss of affinity towards cannabinoid receptors CB1 and CB2.

Nonclassical, Bicyclic-Hydrogenated Cannabinoids
Nonclassical, bicyclic-hydrogenated cannabinoids are exemplified by the paradigm compound CP-55,940 (228, Table 9). This compound acts as a full agonist for both CB1 and CB2 receptors. Compound 229 is obtained by removing the SAH chain from 228 and this leads to the reduction in affinity towards both receptors, CB1 and CB2. Attaching a cyclohexyl group to ring C increases the receptor binding affinity depending on the stereochemistry of the linkage of this group (compound 230 and 231, Table 9).

Nonclassical, Bicyclic-Hydrogenated Cannabinoids
Nonclassical, bicyclic-hydrogenated cannabinoids are exemplified by the paradigm compound CP-55,940 (228, Table 9). This compound acts as a full agonist for both CB1 and CB2 receptors. Compound 229 is obtained by removing the SAH chain from 228 and this leads to the reduction in affinity towards both receptors, CB1 and CB2. Attaching a cyclohexyl group to ring C increases the receptor binding affinity depending on the stereochemistry of the linkage of this group (compound 230 and 231, Table 9).

Nonclassical, Bicyclic-Hydrogenated Cannabinoids
Nonclassical, bicyclic-hydrogenated cannabinoids are exemplified by the paradigm compound CP-55,940 (228, Table 9). This compound acts as a full agonist for both CB1 and CB2 receptors. Compound 229 is obtained by removing the SAH chain from 228 and this leads to the reduction in affinity towards both receptors, CB1 and CB2. Attaching a cyclohexyl group to ring C increases the receptor binding affinity depending on the stereochemistry of the linkage of this group (compound 230 and 231, Table 9).

Nonclassical, Bicyclic-Hydrogenated Cannabinoids
Nonclassical, bicyclic-hydrogenated cannabinoids are exemplified by the paradigm compound CP-55,940 (228, Table 9). This compound acts as a full agonist for both CB1 and CB2 receptors. Compound 229 is obtained by removing the SAH chain from 228 and this leads to the reduction in affinity towards both receptors, CB1 and CB2. Attaching a cyclohexyl group to ring C increases the receptor binding affinity depending on the stereochemistry of the linkage of this group (compound 230 and 231, Table 9).

Nonclassical, Bicyclic-Hydrogenated Cannabinoids
Nonclassical, bicyclic-hydrogenated cannabinoids are exemplified by the paradigm compound CP-55,940 (228, Table 9). This compound acts as a full agonist for both CB1 and CB2 receptors. Compound 229 is obtained by removing the SAH chain from 228 and this leads to the reduction in affinity towards both receptors, CB1 and CB2. Attaching a cyclohexyl group to ring C increases the receptor binding affinity depending on the stereochemistry of the linkage of this group (compound 230 and 231, Table 9).

Nonclassical, Bicyclic-Hydrogenated Cannabinoids
Nonclassical, bicyclic-hydrogenated cannabinoids are exemplified by the paradigm compound CP-55,940 (228, Table 9). This compound acts as a full agonist for both CB1 and CB2 receptors. Compound 229 is obtained by removing the SAH chain from 228 and this leads to the reduction in affinity towards both receptors, CB1 and CB2. Attaching a cyclohexyl group to ring C increases the receptor binding affinity depending on the stereochemistry of the linkage of this group (compound 230 and 231, Table 9).  Table 10) exhibit comparable high calculated binding energies to the CB2 receptor, although the binding energy of the S-HHC epimer (7) was a little lower. The hydrophobic interactions with the amino acid residues of the receptor protein are crucial and they led to equal results for the three cannabinoids. However, for the CB1 receptor, R-HHC (1) and Δ9-THC (35b) displayed similar high calculated binding affinities, while Δ8-THC (36b) and S-HHC (7) bound to this receptor with lower affinity. HHCs (1 and 7) exhibited partial CB1 and CB2 receptor agonist activity similar to Δ9-THC (35b). However, epimer 1 (R-HHC) binds with better affinity (Ki = 15 and 13 nM at CB1 and CB2, respectively) than epimer 7 (S-HHC).
Thapa and co-workers [31,112,113] demonstrated that compounds 232 and 233 are potent angiogenesis inhibitors. They inhibit endothelial and tumor cell growth and lock the secretion of VEGF in cancer cells. Interestingly, these two compounds have poor binding affinities for CB1 and CB2 receptors, showing lower binding energy for both receptors.  (7) was a little lower. The hydrophobic interactions with the amino acid residues of the receptor protein are crucial and they led to equal results for the three cannabinoids. However, for the CB1 receptor, R-HHC (1) and ∆9-THC (35b) displayed similar high calculated binding affinities, while ∆8-THC (36b) and S-HHC (7) bound to this receptor with lower affinity. HHCs (1 and 7) exhibited partial CB1 and CB2 receptor agonist activity similar to ∆9-THC (35b). However, epimer 1 (R-HHC) binds with better affinity (Ki = 15 and 13 nM at CB1 and CB2, respectively) than epimer 7 (S-HHC).
Thapa and co-workers [31,112,113] demonstrated that compounds 232 and 233 are potent angiogenesis inhibitors. They inhibit endothelial and tumor cell growth and lock the secretion of VEGF in cancer cells. Interestingly, these two compounds have poor binding affinities for CB1 and CB2 receptors, showing lower binding energy for both receptors.
Theses in in vitro and in silico studies related to binding affinities of HHC analogs prove how minimal alterations to the HHC scaffold can lead to notable differences in the biological activity of these compounds. Additionally, these results evidence the importance to isolate or of a single diastereomer to study how influential the changes are in the threedimensional structure regarding both toxicology and potency. results for the three cannabinoids. However, for the CB1 receptor, R-HHC (1) and Δ9-THC (35b) displayed similar high calculated binding affinities, while Δ8-THC (36b) and S-HHC (7) bound to this receptor with lower affinity. HHCs (1 and 7) exhibited partial CB1 and CB2 receptor agonist activity similar to Δ9-THC (35b). However, epimer 1 (R-HHC) binds with better affinity (Ki = 15 and 13 nM at CB1 and CB2, respectively) than epimer 7 (S-HHC). Thapa and co-workers [31,112,113] demonstrated that compounds 232 and 233 are potent angiogenesis inhibitors. They inhibit endothelial and tumor cell growth and lock the secretion of VEGF in cancer cells. Interestingly, these two compounds have poor binding affinities for CB1 and CB2 receptors, showing lower binding energy for both receptors. : Alkyl, π-alkyl, π-σ bond, C-H bond, van der Waals. CB 2 : Alkyl, π-alkyl, π-π-T-shaped, π-σ bond 15 9.1 [91,111] results for the three cannabinoids. However, for the CB1 receptor, R-HHC (1) and Δ9-THC (35b) displayed similar high calculated binding affinities, while Δ8-THC (36b) and S-HHC (7) bound to this receptor with lower affinity. HHCs (1 and 7) exhibited partial CB1 and CB2 receptor agonist activity similar to Δ9-THC (35b). However, epimer 1 (R-HHC) binds with better affinity (Ki = 15 and 13 nM at CB1 and CB2, respectively) than epimer 7 (S-HHC). Thapa and co-workers [31,112,113] demonstrated that compounds 232 and 233 are potent angiogenesis inhibitors. They inhibit endothelial and tumor cell growth and lock the secretion of VEGF in cancer cells. Interestingly, these two compounds have poor binding affinities for CB1 and CB2 receptors, showing lower binding energy for both receptors. Table 10. Molecular Docking with D9THC (35b), D8THC (36b), HHC (1 and 7), and HHC analogs (232 and 233) binding with CB1 and CB2 receptor.

Pharmacological and Toxicological Properties of Saturated Cannabinoids
Given the emergence of in vivo studies on the use of saturated cannabinoids in the treatment of various diseases, including cancer [15,[114][115][116][117][118], neurological disorders [64,119,120], and diabetes [121,122], but also the prevalence of the consumption of these compounds [28], there is a crucial need to better comprehend their pharmacology and toxicology. In particular, the role of intrinsic efficacy in abuse-related effects, major metabolites, and adverse effects should be the subject of future study. Very limited information is available on the safety of saturated cannabinoids in humans, and serious health damage is highly likely to occur in those who abuse them. In particular, such information will help public health understanding of the adverse effect profile that differs 232 −6.4 −7.1 ->1000 >100 [31,112,113] Molecules 2023, 28, x FOR PEER REVIEW 51 of 59 232 −6.4 −7.1 ->1000 >100 [31,112,11 3] 233 −5.9 −6.5 ->1000 >100 [31,112,11 3] Theses in in vitro and in silico studies related to binding affinities of HHC analogs prove how minimal alterations to the HHC scaffold can lead to notable differences in the biological activity of these compounds. Additionally, these results evidence the importance to isolate or of a single diastereomer to study how influential the changes are in the three-dimensional structure regarding both toxicology and potency.

Pharmacological and Toxicological Properties of Saturated Cannabinoids
Given the emergence of in vivo studies on the use of saturated cannabinoids in the treatment of various diseases, including cancer [15,[114][115][116][117][118], neurological disorders [64,119,120], and diabetes [121,122], but also the prevalence of the consumption of these compounds [28], there is a crucial need to better comprehend their pharmacology and toxicology. In particular, the role of intrinsic efficacy in abuse-related effects, major metabolites, and adverse effects should be the subject of future study. Very limited information is available on the safety of saturated cannabinoids in humans, and serious health damage is highly likely to occur in those who abuse them. In particular, such information will help public health understanding of the adverse effect profile that differs 233 −5.9 −6.5 ->1000 >100 [31,112,113]

Pharmacological and Toxicological Properties of Saturated Cannabinoids
Given the emergence of in vivo studies on the use of saturated cannabinoids in the treatment of various diseases, including cancer [15,[114][115][116][117][118], neurological disorders [64,119,120], and diabetes [121,122], but also the prevalence of the consumption of these compounds [28], there is a crucial need to better comprehend their pharmacology and toxicology. In particular, the role of intrinsic efficacy in abuse-related effects, major metabolites, and adverse effects should be the subject of future study. Very limited information is available on the safety of saturated cannabinoids in humans, and serious health damage is highly likely to occur in those who abuse them. In particular, such information will help public health understanding of the adverse effect profile that differs from saturated cannabinoids to marijuana [123].

In Vitro Effects of Saturated Cannabinoid Analogs in Pancreatic Cell Lines
We recently reported the preliminary outcomes of the anticancer properties of HHC analogs in four pancreatic cancer cell lines: PANC-1, HPAF-II, AsPc-1, and MIA-PaCa2 [124,125]. Both the (R)-HHC and (S)-HHC epimers equally reduced the proliferation of cancer cells with IC 50 values extending from 10.3 to 27.2 µM. These values are similar to the IC 50 values of the anticancer agents olaparib or veliparib, resulting in more efficient compounds for the specific treatment of pancreatic cancer. Optimization led to novel saturated cannabinoids with greater cytotoxicity towards comparable cell lines [125]. The CCL compounds that were obtained for Colorado Chromatography Lab have exhibited 400-900 nm values against MiaPaCa-2 and PANC-1 cell lines, being over an order of magnitude more potent than Gemcitabine [126]. Although the IC 50 values are lower compared to other active antineoplastic compounds on the market, the treatment of pancreatic cancer is still evolving and the need to produce antineoplastics is pertinent. Continued SAR and analog studies are currently being conducted for our research group to increase bioavailability and increase IC 50 values to lower nanomolar concentrations, with future results potentially supporting our experimental claims.

In Vivo Effects of Saturated Cannabinoid Analogs
CBD and THC have been extensively studied and many in vivo studies related to their anticancer and nausea and pain-relieving activity have been carried out. There are even several FDA-approved human treatments. However, there are very few in vivo studies using saturated cannabinoids and only nabilone (88) has been approved by the FDA to treat nausea and vomiting caused by cancer chemotherapy [127]. Also, preliminary studies propose that nabilone can be used as an acceptable treatment option for severe behavioral problems in adults with intellectual and developmental disabilities [128].
Our research group conducted in vivo studies with CCL compounds to prove the preclinical efficacy of these saturated cannabinoids in a subcutaneous xenograft of pancreatic ductal adenocarcinoma cell lines [126]. These studies indicate that CCL compounds slow down the development of human tumors in a mouse subcutaneous xenograft model, and most intriguingly, demonstrated~50% tumor growth inhibition without significant body weight loss or any unusual signs of toxicity via the oral route (31 mg/kg).
The new and rediscovered cannabinoids have no pre-clinical safety profile performed on them and are being consumed. We executed a pre-clinical assessment on the racemic mixture of HHC [11] and H 4 CBD [129] to provide a preclinical assessment profile for the consumption of these compounds. The analysis of the different cell types revealed varying responses to H 4 CBD and HHC. Lung fibroblasts (NHLF) showed a concentration-dependent reduction in cell viability, with maintained concentrations over 24 h at 6.25-30 µM ensuing in a significant loss of viability. On the contrary, hepatocytes showed a trend of reduced viability at longer exposure times and higher concentrations, but severe cytotoxicity was not observed. This suggests that hepatocytes are less susceptible to the cytotoxic effects of H 4 CBD and HHC compared to NHLF. In the hERG assay, H 4 CBD and HHC did not inhibit the action potentials within cardiomyocytes, indicating no inhibition of ion channels involved in cardiac function.
These findings provide insight into the cytotoxic effects of H 4 CBD and HHC and contribute to establishing research and safety parameters as these compounds continue to gain attention.
Cannazza and coworkers [130] led some in vivo behavioral tests on mice to evaluate the cannabimimetic activity of both HHC diastereomers. These tests judge spontaneous activity, catalepsy, analgesia, and changes in rectal temperature, which are physiological symptoms of THC activity. The outcomes revealed that compound 1 (9R-HHC) extensively altered spontaneous locomotion and pain relief while compound 7 (9S-HHC) had insignificant activity. These discoveries support the in vitro results related to binding affinity to CB1 and CB2 receptors of both diastereomers.
Graziano et al. [14] carried out studies in vivo with both HHC diastereomers displaying effects in the central nervous system, with lower potency than ∆9-THC. Also, this study revealed that 9(R)-HHC is more potent than 9(S)-HHC, suggesting that this diastereomer could lead to a possible addiction potential.

Summary and Outlook
The markets for hydrogenated cannabinoids and related synthetic cannabinoids are rapidly evolving areas with relatively limited information currently available. This review summarizes the discovery, novel synthetic pathways, and pharmacology studies of classical, non-classical, and hybrid hydrogenated cannabinoids, discussing the most critical point of view in this area. This is harmonized with a summary and comparison of the cannabinoid receptor affinities of various classical, hybrid, and non-classical saturated cannabinoids. A discussion of structure-activity relationships with the four different pharmacophores found in the cannabinoid scaffold is added to this review.
Saturated cannabinoid-based therapies like nabilone suffer from undesirable pharmacological properties including poor bioavailability, the unpredictable onset/offset of action, and detoxification. The clear medical need for novel cannabinoid-based medications has encouraged us to pursue this review. We believe the design and development of novel hydrogenated cannabinoids should address the quest for new selective antagonist-based cannabinoids for CB2 receptors with improved drug ability, i.e., improved oral availability, a predictable time course of action, and controllable detoxification. The design of new CB2-selective hydrogenated THC analogs should have little or no affinity for the CB1 receptor, thus eliminating the risk of central CB1-mediated psychotropic effects.
Furthermore, the input of an azido, isothiocyanate, and cyano-moiety at diverse tactical positions within these nonclassical-hybrid hydrogenated cannabinoids and the emergence of covalent bonds with different amino acid residues on the CB1 and CB2 receptors allow for a more comprehensive searching of the stereochemical features of the receptor active sites.
The cannabinoid-based research should focus on accomplishing more efficient enantioselective routes to furnish novel synthetic and highly enantiopure-saturated nonclassical and hybrid cannabinoids at the disposal of chemists. Many more exclusive ligands can be minded and explored for their pharmacological activity. The accessibility of the functionalized bi-and tricyclic cannabinoid skeleton will facilitate the scanning of the CB1 and CB2 receptors. A better comprehension of the receptor binding site may make it possible to project cannabinoids with controlled selectivity and affinity for CB1, CB2, or both cannabinoid receptors to potentially support in the selective handling of the endocannabinoid system.
The limitation of the study of saturated cannabinoids is that most of the articles do not offer a multiparty vision between the challenges of organic synthesis, medicinal chemistry, and toxicology of these compounds, which play an important role in the cannabinoid research.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.