Drug-like chemical space based on Lipinski’s rule of five for drug candidates is explored to improve its pharmacokinetic properties. (36) Five physiochemical descriptors covered under Lipinski’s rule of five are shown for all of the metabolites along with reference psychoactive compounds in Figures 1 6 . The molecular weights of the 468 metabolites from, its smoke, and other phytocannabinoids are shown in Figure 1

Figure 1. Molecular weights of metabolites from C. sativa , its smoke, and other phytocannabinoids. Orange circle solid represents metabolite, yellow circle solid reoresents psychoactive reference compound, green circle solid represents endocannabinoid, black circle solid represents THC, and black and red lines represent Lipinski’s and the central nervous system (CNS) cutoff, respectively.

With Lipinski’s rule of five, only 13 metabolites violated the cutoff scale of 500 and all of the Schedule I−IV drugs were well below the Lipinski’s cutoff (black line) for molecular weight. Pajouhesh et al. (37) in the analysis of physiochemical descriptors for drugs, which include CNS acting drugs, reported the new cutoff values for physiochemical descriptors for CNS-targeted drugs. The CNS cutoff (red line) for molecular weight was 310, and when it was applied to the 468 metabolites, it showed a significant number of outliers, which include 53% of Schedule I−IV substances. Endocannabinoids except oleamide, which are natural agonists for cannabinoid receptors, display molecular weight above the CNS cutoff value. It is important to mention that THC has the molecular weight (314) that is closer to CNS cutoff having a psychoactive property in

Figure 2. M log P of metabolites from C. sativa , its smoke, and other phytocannabinoids. Orange circle solid represents metabolite, yellow circle solid represents psychoactive reference compound, green circle solid represents endocannabinoid, black circle solid represents THC, and black and red lines represent Lipinski’s and the CNS cutoff, respectively.

Most of the metabolites along with Schedule I−IV drugs obey Lipinski’s cutoff and showed deviation from the CNS drug cutoff value of 2.08. Cannabinoids from C. sativa, phytocannabinoids, and the volatile products derived from C. sativa showed moderate hydrophobicity.

Total polar surface area (TPSA) controls the absorption property of the drugs, (38−40) and Lipinski’s cutoff is 140 Å. Any drug above this value shows poor absorption into the intestines. TPSA for all of the metabolites, endocannabinoids, and Schedule I−IV substances is shown in Figure 3

Figure 3. TPSA of metabolites from C. sativa , its smoke, and other phytocannabinoids. Orange circle solid represents metabolite, yellow circle solid represents psychoactive reference compound, green circle solid represents endocannabinoid, black circle solid represents THC, and black and red lines represent Lipinski’s and the CNS cutoff, respectively.

The cutoff value of TPSA for CNS drugs is 70 Å2, and surprisingly the majority of metabolites from C. sativa have TPSA lower than 70 Å2. THC showed a TPSA value of 29 Å2, and the highest TPSA value among endocannabinoids is 69 Å2. Cannabidiol (CBD), which does not have a psychoactive property, showed a TPSA value of 78 Å2, and lower TPSA is pronounced in the case of cannabinoids having a psychoactive property. TPSA may be one of the best filters to identity the metabolites from cannabinoids having psychoactive properties.

H-bond donor and H-bond acceptor properties determine the mode and strength of ligand–receptor interaction through hydrogen bonds. As per Lipinski’s rule of five, the permissible number of H-bond donors is 5 and H-bond acceptors is 10 for a successful drug. However, in the case of CNS drugs, the cutoff is reduced to 1.5 for a H-bond donor and 4.32 for a H-bond acceptor. As shown in Figure 4 a, the majority of metabolites and endocannabinoids have maximum of 3 H-bond donors. In the case of Schedule I−IV substances, 95% are shown to have only 2 H-bond donors.

Figure 4. (a) H-bond donors of metabolites from C. sativa , its smoke, and other phytocannabinoids. Orange circle solid represents metabolite, yellow circle solid represents psychoactive reference compound, green circle solid represents endocannabinoid, black circle solid represents THC, and black and red lines represent Lipinski’s and the CNS cutoff, respectively. (b) H-bond acceptor of metabolites from C. sativa , its smoke, and other phytocannabinoids. (Orange circle solid) metabolite, (yellow circle solid) psychoactive reference compound, (green circle solid) endocannabinoid, (black circle solid) THC, black and red lines represent Lipinski’s and the CNS cutoff, respectively.

It is clear that CNS drugs and Schedule I−IV substances interact with their target receptors through hydrophobic interaction and less dependent on the H-bond network. THC showed only one H-bond donor and CBD had three H-bond donors. The CDK molecular descriptor tool calculates H-bond acceptors for the molecule when an oxygen or a nitrogen in the molecule has formal charge equivalent to or less than zero (formal charge ≤0). Figure 4 b shows that the majority of cannabinoids, endocannabinoids, and the Schedule I−IV substances showed four H-bond acceptors.

Effect of the H-bond acceptor on drug efficacy is less pronounced from Lipinski’s point of view, and Figure 4 b shows that Schedule I−IV substances having the H-bond acceptor value between 5 and 7 are able to penetrate the blood–brain barrier (BBB) and exhibit excellent psychoactive activity.

The number of rotatable bonds affects the molecular conformational freedom, and a molecule having less number of rotatable bonds has structural rigidity. It is important to freeze the bioactive conformation, and it is achieved by reducing the number of rotatable bonds. (41) Figure 5 shows the number of rotatable bonds for metabolites from cannabis, endocannabinoids, and the Schedule I−IV substances.

Figure 5. Number of rotatable bonds of metabolites from C. sativa , its smoke, and other phytocannabinoids. Orange circle solid represents metabolite, yellow circle solid represents psychoactive reference compound, green circle solid represents endocannabinoid, black circle solid represents THC, and black and red lines represent Lipinski’s and the CNS cutoff, respectively.

Compliance with Lipinski’s rule of five with a number of rotatable bonds (<10) is high for most cannabis metabolites and the Schedule I−IV substances. All of the endocannabinoids violate Lipinski’s rule with regard to the number of rotatable bonds being in the range of 15–19. It is to be noted that endocannabinoids are lipid moieties having a high number of freely rotatable bonds, they are synthesized and metabolized in the brain, and moreover they do not have to cross the blood–brain barrier.

Machine learning methods are recent tools in identifying the receptor targets based on structure- and ligand-dependent approaches in the drug discovery process. (42) In this study, four classifiers, namely, multilayer perceptron, naïve-Bayes, support vector machine, and-Nearest Neighbor were used to identify opiate- and THC/endocannabinoid-like molecules. Initially, the results of cross-validation performed on the reference candidates consisting of Schedule I−IV substances, THC endocannabinoids agonists, and negative controls provided the results shown in Table 1

The performance of four classifiers for three types of data was assessed based on their accuracy, sensitivity, specificity, ROC area, and F-measure values. Accuracy quantifies the efficiency of each classifier to predict the true values. The Matthews correlation coefficient (MCC) allows one to judge the performance of a given classifier. MCC ranges from −1 to +1, where −1 indicates that it is a wrong binary classifier and +1 is the indication of the correct classifier. High positive values of ROC and PRC indicate that the classifier shows high performance for a given data set. Accuracy ranged from 85 to 99% for different classifiers with three data types. Sensitivity and specificity quantify the proportion of true positives and negatives, respectively. Both sensitivity and specificity ranged from 77 to 100%, where 77% was shown by the NB classifier with CDK–ADMET hybrid data type.

As a next step of filtering the psychoactive cannabinoid candidates, nonmetric multidimensional scaling was performed on 330 molecular candidates obtained from machine learning methods. Multidimensional scaling (MDS) is a valuable tool to obtain a degree of similarity among the members of a group in a given data set. (44−46) MDS has two methods, that is, metric and nonmetric MDS. Metric MDS measures similarity-based interpoint distances between the members of the group and nonmetric MDS operates by the relative ordering of similarity in an ensemble of data points. Nonmetric MDS has the advantage over metric MDS as the magnitude of similarity of input data points is unreliable or sometimes difficult to measure with high accuracy. MDS has been applied to analyze the patterns of gene expression (47) and evolutionary pathways of GPCR. (48) Nonmetric MDS was performed on 330 candidates using physiochemical molecular descriptors and later using ADMET descriptors. In the NMDS map, the outliers were excluded and the remaining candidates were retained for further analysis. NMDS on physiochemical descriptors resulted in 165 candidates ( Supplement 2 ), and the subsequent NMDS using clinical descriptors provided 112 candidates ( Supplement 1 ). NMDS maps for CDK and ADMET are shown in Figures 6 and 7

The list of candidates classified as a drug by all four classifiers from three data types is given in Supplement 1 . The four classifiers eliminated 138 candidates from the original database based on the comparison of predicted physiochemical and clinical descriptors of Schedule I−IV drugs, endocannabinoid agonists, and negative controls. In the remaining 330 candidates, about 41% of the candidates in the list belong to volatile metabolites, and these volatile compounds gain importance, as smoking is the most common route of consumption of cannabis products. Smoking is the quickest way to have a rapid onset of psychoactive effect due to the high bioavailability of THC in the systemic circulation. Interestingly, three alkanes, namely, 3-methyl heptane, 4-methyl decane, and nonane were also found in the volatile mixture of cannabis smoke. Alkanes possess anesthetic effect, and the anesthetic potency decreases with increasing chain; moreover, no anesthetic effect is observed from-undecane (C-11). (43)

Overfitting of data is diagnosed on the fact that whenever there is overfitting, the accuracy drastically reduces from training to test sets. Our results showed that for CDK data type, accuracy was significantly reduced for NB and kNN classifiers from training to test sets. In the case of CDK–NB combination, the training set was observed to be 98.64% and it was reduced to 57% for the test set. Similarly, in the case of CDK–kNN combination, the accuracy of 100% observed for the training set was reduced to 57% for the test set. It can be concluded that NB and kNN classifiers overfit the data for CDK data type and hence these two classifiers were not included for analysis involving CDK data type.

Accuracy results in the training, cross-validation, and test for three data types (CDK, CDK–ADMET, and ADMET) using four classifiers on three unique datasets on reference candidates (training, validation, and test sets) are given in Table 2 a–c.

Structure–Activity Relationship of Cannabinoids

A common scaffold of THC was compared by three-dimensional (3D) alignment with every candidate in the group of 112 members from NM-MDS analysis, and the score obtained for 3D alignment was listed from the highest to lowest value. The minimum cutoff value of 0.5 was fixed for 3D alignment, as the cannabidiol having less psychoactive character compared to THC has a homology score of 0.558, and hence it is taken as the baseline for assessing the structure–activity relationship. The candidates having 3D homology till 0.5 were selected for study on the structure–activity relationship and scaffold homology of each metabolite with THC, and changes in pharmacophores of each metabolite in comparison to THC and their corresponding chemical structures are given in Tables 3A and 3B . In addition, 3D-aligned structures of each metabolite with THC are provided in Supplement 4

Table 3A. Changes in Pharmacophores of the THC Scaffold Leading to Various Cannabinoid Metabolites Found in C. sativa sl. no. cannabinoid scaffold homology To THC 1-methyl cyclohexene benzopyran phenolic group C-3-side chain any other cause 1 8a-hydroxy-delta-9-tetrahydrocannabinol 0.95 OH at 8th position 2 delta-9-tetrahydrocannabinol-C-4 0.945 one CH 2 less to THC 3 delta-8-tetrahydrocannabinolic acid 0.907 COOH at position 2 low BBB 4 9β,10β-epoxyhexahydrocannabinol 0.859 loss of unsaturation at position 9 and converted to the epoxide ring 5 9α,10α-tetrahydrocannabinol epoxide 0.859 loss of unsaturation at position 9 and converted to the epoxide ring 6 7,8-dihydrocannabinol 0.825 additional double bond at position 7 7 9,10-anhydrocannabitriol 0.825 epoxide ring at position 9 and a double bond at position 10a 8 10-hydroxy-9-oxo-delta-8-tetrahydrocannabinol 0.802 double bond at position 8 and carbonyl at position 9 and OH group at position 10 9 10-oxo-delta-6a-tetrahydrocannabinol (OTHC) 0.744 carbonyl at position 10 double bond at position 6a 10 cannabichromanone (CBCF) 0.73 ring opens carbonyl at position 10a 11 delta-8-tetrahydrocannabinol (Δ8-THC) 0.722 double bond at position 8 instead of 9 12 isocannabitrol 0.709 OH group at positions 8, 9 and ethylene group at position 10 13 10α-hydroxy-delta-9,11-hexahydrocannabinol 0.706 ethylene group at position 9 and OH group at position 8 14 compound-3 0.692 ring opens, OH group at position 7 and a double bond at position 9 oxidized to aldehyde 15 delta-9-tetrahydrocannabinolic acid B (THCA-B) 0.681 COOH group at position 4 low BBB 16 delta-9-cis-tetrahydrocannabivarin 0.678 cis isomer at position 9 propyl group instead of pentyl 17 compound-2 0.673 ring opens, double bond at C-9–C-10 oxidized to ketone and aldehyde 18 cannabichromaonone D 0.654 ring fused with both phenolic and pyran rings 19 compound-4 0.654 ring opens and fused with a phenolic group keto group at position 10a 20 delta-9-nor-tetrahydrocannabinol 0.647 ethylene group at position C-8 butyl group at instead of pentyl 21 2-formyl-delta-9-tetrahydrocannabinol 0.645 ethylene group at position C-8 formyl group at position 2 22 cannabicyclolic acid (CBLA) 0.639 ring converted to fused cyclopentyl and cyclobutyl groups COOH group at position 2 low BBB 23 8-oxo-delta-9-tetrahydrocannabinol 0.638 oxo group at position 8 24 hexahydrocannabinol 0.636 double bond at C-9 is reduced 25 bis-nor-cannabitriol 0.632 double bond at C-9 reduced, OH group at C-9 added, vinyl alcohol is added to position C-10 26 cannabinol (CBN) 0.63 converted to benzene ring 27 6a-R-cannabichromanone B 0.627 ring opens keto group at position 10a and methyl 3-hydroxy propenyl ketone is added to position 6a low BBB 28 7-hydroxy cannabinol 0.618 ring converted to hydroxy benzene 29 7-hydroxy cannabichromane 0.616 ring opens 2-methyl-2-pentene attached to position 6 30 delta-7-trans-isotetrahydrocannabinol 0.614 ring opens ring opens 31 10-ethoxy-9-hydroxy-delta-6a-tetrahydrocannabinol 0.608 ethoxy group at position 10 and OH group at position 9 32 delta-9-tetrahydrocannabinolic acid A (THCA-A) 0.608 COOH at position 2 low BBB 33 cannabinol methyl ether (CBNM) 0.592 same as cannabinol 34 delta-9-nor-tetrahydrocannbinolic acid 0.592 ethylene group at position 8 COOH at position 2 butyl group at instead of pentyl 35 delta-9-tetrahydrocannabiorcol (THC-C1) 0.587 methyl group instead of pentyl 36 anhydrocannabimovone 0.586 pyran is replaced by tetrahydrofuran 37 10-O-ethyl bis-nor cannabitriol 0.584 double bond at C-9 reduced, OH group at C-9 added, ethyl vinyl ether at position 10 38 bis-nor-cannabichromanone 0.574 ring opens propyl instead of pentyl group 39 cannabielsoin (CBE) 0.554 pyran is replaced by tetrahydrofuran 40 delta-9-tetrahydrocannabinolic acid-C-4 (THCA-C-4) 0.554 COOH at position 2 butyl instead of pentyl group 41 cannabicyclovarin (CBLV) 0.547 ring converted to fused cyclopentyl and cyclobutyl groups propyl instead of pentyl group 42 delta-9-tetrahydrocannabiorcolic acid (THCA-C1) 0.547 COOH at position 2 methyl group instead of pentyl 43 O-propyl-cannabidiol 0.547 ring opens, propyl group at OH group 44 cannabidiol (CBD) 0.539 ring opens 45 3-hydroxy-delta-4,5-cannabichromene 0.536 ring opens methyl at position 7 is substituted by 3-hydroxy-pentene group 46 3,4,5,6-tetrahydro-7-hydroxy-a,a-2-trimethyl-9-n-propyl-2,6-methano-2H-1-benzoxocin-5-methanol 0.535 ring opens cyclopentyl group attached to ortho and OH groups 47 cannabicoumaronone 0.53 4,5-dehydrofuran group fused with benzopyran and phenolic groups 48 delta-7-cis-isotetrahydrocannabivarin 0.528 ring opens cyclopentyl group attached to ortho and OH groups propyl instead of pentyl group 49 cannabidivarin (CBDV) 0.522 ring opens propyl instead of pentyl group 50 cannabinodivarin (CBVD) 0.522 replaced by benzene ring ring opens propyl instead of pentyl group 51 cannabiglendol 0.514 replaced by tetrahydrofuran group propyl instead of pentyl group 52 cannabidiol monomethyl ether (CBDM) 0.508 ring opens, methyl group at OH group 53 4-acetoxy cannabichromene 0.501 ring opens methyl at position 7 is substituted by 2-methyl-pentene group acetoxy group at position 4 54 7-R-cannabicoumarononic acid 0.5 ring opens 4,5-dehydrofuran group fused with benzopyran and phenolic groups COOH at position 2

Table 3B. Chemical Structures of 54 Metabolites Having High 3D Homology with THC

a-hydroxy-delta-9-tetrahydrocannabinol. As shown in P value, thereby increasing membrane solubility and favorable interaction energy with the receptor. Delta-9-tetrahydrocannabinol-C-4 (cis-tetrahydrocannabivarin ( The metabolite having the highest scaffold homology (0.95) is 8-hydroxy-delta-9-tetrahydrocannabinol. As shown in Supplement 4-1 , it has an additional hydroxy moiety at 8th position in the cyclohexene ring and it makes the molecule more hydrophilic than THC. THC is highly lipophilic and the hydrophobic regions such as cyclohexene ring, C-5 chain on the phenolic moiety, and pyran ring interact with membrane lipid hydrophobic acyl chains and hydrophobic amino acids in transmembrane helices VI and VII of GPCR as reported in the literature. (49) Introducing an OH group at 8th position on the cyclohexene ring reduces the partition distribution into the membrane, and it causes unfavorable energy of the OH group interacting with hydrophobic membrane lipids and the amino acids in the receptor. Liposome-mediated delivery and masking the OH group by alkylating with a methyl or an ethyl group may increase the logvalue, thereby increasing membrane solubility and favorable interaction energy with the receptor. Delta-9-tetrahydrocannabinol-C-4 ( Supplement 4-2 ) has homology score of 0.945 and C-5 chain at position 3 of the phenol group is reduced to C-4 and any decrease in hydrophobic chain length reduces the affinity with the receptor and potency, as the hydrophobic interaction of C-5 chain with membrane lipids and receptor is vital for receptor activation. Delta-9--tetrahydrocannabivarin ( Supplement 4-16 ), apart from cis configurational change at 9th position in cyclohexane ring, has a propyl group instead of pentyl at C-3 position of the phenolic group, which reduces its affinity for the receptor. Delta-9-tetrahydrocannabiorcol (THC-C1) ( Supplement 4-35 ) has a methyl group instead of pentyl at the C-3 position of the phenolic group, and it reduces the affinity for the CB1 receptor significantly.

Epoxide ring derivatives such as 9β,10β-epoxyhexahydrocannabinol ( Supplement 4-4 ), 9α,10α-tetrahydrocannabinol epoxide ( Supplement 4-5 ), and 9,10-anhydrocannabitriol ( Supplement 4-7 ) have epoxide rings at positions 8–9 in the cyclohexene ring, this epoxide ring is highly susceptible to hydrolysis under acidic conditions existing in the stomach, and the end product 8,9-dihydroxy THC is more polar than THC and hence it may have lower capacity to cross the blood–brain barrier and partition into the membranes is reduced similar to 8-hydroxy THC.

a-tetrahydrocannabinol ( The metabolite 7,8-dihydrocannabinol ( Supplement 4-6 ) has an additional double bond at 10–6a position in the cyclohexene ring and makes it more reactive to undergo metabolism in the liver. Otherwise, it may be expected to have bioactivity equivalent to THC. In 10-hydroxy-9-oxo-delta-8-tetrahydrocannabinol ( Supplement 4-8 ) and 10-oxo-delta-6-tetrahydrocannabinol ( Supplement 4-9 ) (OTHC), additional OH and carbonyl groups at positions 10a and 9 in the cyclohexene ring make the overall molecule more polar having one more H-bond donor and H-bond acceptor. The extra OH and carbonyl groups may result in dimerization by head-to-tail mode of interaction. An increase in polarity reduces the partition distribution into the membrane to interact with GPCR.

Delta-8-THC ( Supplement 4-11 ) is an isomer of THC, which differs only in the position of double bond in the cyclohexene ring. Delta-8-THC is less psychoactive than delta-9-THC, and it may be due to low solubility of delta-8-THC in water. (50) Isocannbitrol ( Supplement 4-12 ) having two additional OH groups at 8, 9 positions in the cyclohexene ring makes it highly hydrophilic, and similar to 8-hydroxy-THC, isocannbitrol may have lower partition distribution into the membranes for receptor activation. Similarly, 10-hydroxy-delta-9,11-hexahydrocannabinol ( Supplement 4-13 ) has an extra OH group in the cyclohexene ring and is more polar than delta-9-THC.

Delta-9-nor-tetrahydrocannabinol ( Supplement 4-20 ) has an ethylene group in the cyclohexene group and the alkyl chain at C-3 of the phenolic group is reduced to butyl instead of pentyl group. Dramatic reduction in the scaffold homology (0.647) with the introduction of the ethylene group at position 8 of the cyclohexene group generates steric hindrance, and reduction in chain length at the C-3 position of the phenolic group decreases the affinity for the receptor. In 2-formyl-delta-9-tetrahydrocannabinol ( Supplement 4-21 ), the formyl group at position 2 in the phenolic group may form intra or intermolecular H-bonding with OH group and may lead to dimerization. As the phenolic OH group may be engaged intra- or intermolecularly in 2-formyl-delta-9-tetrahydrocannabinol, its involvement with receptor activation may be affected. In addition, the ethylene group at position 8 of the cyclohexene ring may generate steric effects similar to delta-9-nor-tetrahydrocannabinol. With 8-oxo-delta-9-tetrahydrocannabinol ( Supplement 4-23 ), the introduction of the oxo group increases the polarity and hence it reduces the partition distribution into the membrane to interact with the receptor.

Hexahydrocannabinol ( Supplement 4-24 ) has a reduced cyclohexane ring instead of a cyclohexene ring, which alters the orientation of this ring inside the membrane on receptor interaction due to conformational flexibility.

In cannabinol ( Supplement 4-26 ), 7-hydroxy cannabinol ( Supplement 4-28 ), and cannabinol methyl ether ( Supplement 4-33 ), replacement of the nonplanar cyclohexene ring with a benzene ring abolishes the bioactivity, but its affinity for receptor increases, leading to the antagonistic mode of interaction with GPCR. The aromatic ring in cannabinol has π-electron cloud, which may interact with positively charged amino acid Lys or Arg due to cation−π interaction, resulting in deviation from bioactive conformation. Cation−π binding in cannabinol and 7-hydroxy cannabinol renders a stronger affinity for the receptor.

a-tetrahydrocannabinol (O-Ethyl bis-nor cannabitriol ( Bis-nor-cannabitrol ( Supplement 4-25 ) and 10-ethoxy-9-hydroxy-delta-6-tetrahydrocannabinol ( Supplement 4-31 ) have hydroxyl groups at positions 9 and 10 in the cyclohexene ring, which makes it highly hydrophilic and hence its partition distribution into the membrane reduces significantly. 10--Ethyl bis-nor cannabitriol ( Supplement 4-37 ) has a hydroxyl group at position 9 in the cyclohexyl ring, which makes it more hydrophilic, and reducing the chain length from a pentyl to a propyl group at the C-3 position of the phenolic group leads to low partition distribution into the membrane and reduces the affinity for the CB1 receptor.

a-R-cannabichromanone B ( The five metabolites from cannabis such as compound-2 ( Supplement 4-17 ), compound-3 ( Supplement 4-14 ), 6-cannabichromanone B ( Supplement 4-27 ), 7-hydroxy cannabichromane ( Supplement 4-29 ), and bis-nor-cannabichromanone ( Supplement 4-38 ) lack the cyclohexene ring and substituted ketones occupy position 3 in the benzopyran ring. As per the structure–activity relationship rules of cannabinoids, a nonplanar cyclohexene ring is important but not essential for bioactivity. When the nonplanar cyclohexene ring is replaced by a bulky substituent attached to positions 3 and 4, still the molecules can show bioactivity. However, in the above five metabolites, no bulky substitution is attached to occupy positions 3 and 4 in the benzopyran ring and hence all of these five metabolites may not show bioactivity as THC.

Cannabichromanone ( Supplement 4-10 ) has a fused tetrahydrofuran ring connecting phenolic OH and the benzopyran ring. It replaces the cyclohexene ring, and the 2-butanone group is attached to the C-3 position of the benzopyran ring. It is more nonpolar than THC, and the missing OH moiety at the phenolic group may reduce the favorable interaction energy, thereby reducing the affinity for the CB1 receptor.

trans-isotetrahydrocannabinol ( In the case of delta-7--isotetrahydrocannabinol ( Supplement 4-30 ), the benzopyran ring is broken and as per SAR rules, the loss of benzopyran ring results in the absence of any bioactivity. Cannabichromanone D ( Supplement 4-18 ) has a ring fused at position 3 of the benzopyran ring and the OH group of the phenolic group. It also has a carbonyl group at position 4 of the benzopyran ring. SAR rules of cannabinoids state that even with substitution of the hydroxyl group of phenolic pharmacophore, the molecule retains bioactivity. Hence, cannabichromanone D may show bioactivity, as three out of four pharmacophores may be enough to elicit receptor activation.

In all three metabolites, anhydrocannabimovone ( Supplement 4-36 ), cannabielsoin ( Supplement 4-39 ) (CBE), and cannabiglendol ( Supplement 4-51 ), the 6-membered benzopyran ring is replaced by a 5-membered tetrahydrofuran ring. As the benzopyran ring is essential for bioactivity, its replacement may result in loss of bioactivity.

cis-isotetrahydrocannabivarin ( Delta-7--isotetrahydrocannabivarin ( Supplement 4-48 ) has bulky fused cyclohexyl and cyclopropyl groups that are attached to phenolic OH and C-6 position. The missing pharmacophore, benzopyran group, and propyl group at C-3 of the phenolic group instead of the pentyl group that reduces the affinity for the receptor and leads to complete loss of bioactivity.

a,a-2-trimethyl-9-n-propyl-2,6-methano-2H-1-benzoxocin-5-methanol ( In the 3,4,5,6-tetrahydro-7-hydroxy--2-trimethyl-9--propyl-2,6-methano-2-1-benzoxocin-5-methanol ( Supplement 4-46 ), the absence of an important pharmacophore benzopyran ring results in loss of bioactivity. In addition, the pentyl group at the C-3 position of the phenolic group is replaced by a propyl group, and it reduces the affinity for the CB1 receptor.

Cannabicyclovarin (CBLV) ( Supplement 4-41 ) has bulky fused cyclopentyl and cyclobutyl groups on the benzopyran ring, it is well tolerated for replacement of the cyclohexene group, and it has a propyl group at C-3 of the phenolic ring, which reduces the affinity for the CB1 receptor.

In 3-hydroxy-delta-4,5-cannabichromene ( Supplement 4-45 ) and 4-acetoxy cannabichromene ( Supplement 4-53 ), the basic cannabichromene has a missing cyclohexene ring, and the absence of bulky groups at 3, 4 positions in the benzopyran group makes it biologically inactive. Moreover, in the case of 4-acetoxy cannabichromene, it can undergo hydrolysis in the stomach giving rise to a hydrophilic 4-hydroxy phenolic group derivative. The hydroxyl group in 3-hydroxy-delta-4,5-cannabichromene and post-metabolic 4-acetoxy cannabichromene may have reduced partition into the membrane for receptor activation.

In cannabidivarin ( Supplement 4-50 ) and cannabinodivarin ( Supplement 4-49 ), the benzopyran ring is opened and it is similar to cannabidiol. The difference between cannabidivarin and cannabidiol is that the pentyl group is present at C-3 of the phenolic group in cannabidiol, and it is replaced by a propyl group in cannabidivarin. Hence, cannabidivarin is expected to have less affinity for the CB1 receptor than cannabidiol. Cannabidivarin resembles cannabinol, in which both have benzene rings instead of the cyclohexene ring. They differ with respect to the benzopyran and pentyl groups at C-3 of phenolic pharmacophore.